Devices, systems and methods for inhibiting invasion and metastases of cancer

ABSTRACT

The invention generally relates to a microfluidic platforms or “chips” for testing and understanding cancer, and, more specifically, for understanding the factors that contribute to cancer invading tissues and causing metastases. Tumor cells are grown on microfluidic devices with other non-cancerous tissues under conditions that simulate tumor invasion. The interaction with immune cells can be tested to inhibit this activity by linking a cancer chip to a lymph chip.

FIELD OF THE INVENTION

The invention generally relates to a microfluidic platforms or “chips”for testing and understanding cancer, and, more specifically, forunderstanding the factors that contribute to cancer invading tissues andcausing metastases. Tumor cells are grown on microfluidic devices withother non-cancerous tissues under conditions that simulate tumorinvasion. The interaction with immune cells can be tested to inhibitthis activity by linking a cancer chip to a lymph chip.

BACKGROUND

Success treating particular cancers is also hampered by the fact thatthe cancer is well-advanced by the time it is diagnosed. Metastatictumors in the lungs are cancers that developed at other places in thebody (or other parts of the lungs) and spread through the bloodstream orlymphatic system to the lungs. It is different than lung cancer thatstarts in the lungs. Melanoma is but one of many cancers that canmetastasize to the lungs. A cure is unlikely in most cases of cancersthat have spread to the lungs. But the outlook depends on the underlyingcancer. Some cancers, such as lymphoma, are very treatable and evencurable. In general, it is rare for someone to live more than 5 yearswith metastatic cancer to the lungs.

In sheer numbers, colon cancer is even a bigger killer. With 655,000deaths worldwide per year, it is the third most common form of cancerand the second leading cause of cancer-related death in the Westernworld. When detected late, surgery may be of no use. For example, 20% ofpatients present with metastatic (stage 1 V) colorectal cancer at thetime of diagnosis, and only 25% of this group will have isolated livermetastasis that is potentially resectable. Radiation is not routinelyused since it can cause radiation enteritis. Chemotherapy is often usedpost-surgery as adjunct therapy. However, the use of chemotherapeuticsis complicated by the fact that colon cancer is often found in theelderly, who do not respond well to aggressive chemotherapy.

Breast cancer is the most common malignancy and the second leading causeof cancer death in women. In over 60% of localized breast cancer cases,histologic evidence of tumor spread to surrounding tissue is found.Patients diagnosed with invasive ductal carcinoma, the most commonbreast cancer, have a lower 10-year survival rate. About 30% of newlydiagnosed breast cancer patients have positive lymph nodes and muchpoorer outcomes.

What is needed are better compounds and methods for treating cancer,including advanced cancer and metastatic disease.

SUMMARY OF THE INVENTION

The invention generally relates to a microfluidic platforms or “chips”for testing and understanding cancer, and, more specifically, forunderstanding the factors that contribute to cancer invading tissues andcausing metastases. Tumor cells are grown on microfluidic devices withother non-cancerous tissues under conditions that simulate or supporttumor invasion. The interaction with immune cells can be tested toinhibit this activity by linking a cancer chip to another chip, e.g. alymph chip, a bone marrow chip, a liver chip, etc. The interaction withcirculating immune cells recruited to the tumor site will be enabled toallow testing of immunomodulatory agents. The interaction withcirculating immune cells can be tested to confirm immune surveillance(or the lack thereof) and provide a platform for testing ofimmunotherapeutics. Indeed, these microfluidic platforms can increaseour understanding of tumor growth and all the other aspects of cancer,including but not limited to, the factors that contribute to cancerrelated angiogenesis, the role of ECM on this process, resistance toimmune surveillance, and expansion to other organs underlying thedevelopment of metastatic disease.

Tumor cells are grown on microfluidic devices with other non-canceroustissues under conditions that simulate tumor invasion. In oneembodiment, tumor cells from a biopsy are assessed for their metastaticpotential by seeding them on one or more layers of living cells (e.g.epithelial cells) in a microfluidic device and determining whether theyinvade said one or more layers. The interaction with immune cells can betested to inhibit this activity by, among other things, linking a cancerchip to a lymph chip. In one embodiment, the lymph chip is populated bycells from a lymph node.

In one embodiment, the present invention contemplates a microfluidicdevice comprising: a body having a microchannel (optionally locatedcentrally) therein; and an at least partially porous membrane positionedwithin the microchannel (and optionally along a plane), the membraneconfigured to separate the microchannel to form a first microchannel anda second microchannel, the membrane comprising a top surface and abottom surface, said a) top surface comprises a first layer comprisingliving stromal cells, a second layer positioned on top of said firstlayer and comprising living epithelial cells (or the epithelial cellscan be placed directly on the membrane with no stromal layer), andliving tumor cells in contact with said epithelial cells (or in closeproximity), said b) bottom surface comprising living endothelial cells.In one embodiment, said membrane is coated with at least one attachmentmolecule that supports adhesion of a plurality of living cells. In oneembodiment said first and second microchannels comprise fluid (e.g.culture media, blood, lymph, serum, plasma, etc.). In one embodiment,said tumor cells are from a biopsy. In one embodiment, said tumor cellsare human tumor cells. In one embodiment, said tumor cells are also incontact with at least one type of immune cell (or in close proximity).In one embodiment, said tumor cells are in contact with lymphocytes (orin close proximity). In one embodiment, said tumor cells are in contactwith T cells (or in close proximity). In one embodiment, said T cellsare primed T cells (e.g. T cells that have been exposed to antigen, orone or more cytokines, or to cancer cells, or otherwise activated). Inone embodiment, said tumor cells are in contact with activated dendriticcells (or in close proximity).

In yet another embodiment, the present invention contemplates a systemcomprising first and second microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a bodyhaving a microchannel (optionally located centrally) therein; and an atleast partially porous membrane positioned within the microchannel (andoptionally along a plane), the membrane configured to separate themicrochannel to Ruin a first microchannel and a second microchannel, themembrane comprising a top surface and a bottom surface, said i) topsurface comprises a first layer comprising living stromal cells, asecond layer positioned on top of said first layer and comprising livingepithelial cells, and living tumor cells in contact with said epithelialcells (or in close proximity), said ii) bottom surface comprising livingendothelial cells; said b) second microfluidic device comprising immunecells. In one embodiment, said membrane is coated with at least oneattachment molecule that supports adhesion of a plurality of livingcells. In one embodiment, said first and second microchannels of saidfirst microfluidic device comprise fluid. In one embodiment, said tumorcells are from a biopsy. In one embodiment, said tumor cells are humantumor cells. In one embodiment, said tumor cells are also in contactwith at least one type of immune cell (or in close proximity). In oneembodiment, said tumor cells are in contact with lymphocytes (or inclose proximity). In one embodiment, said tumor cells are in contactwith T cells (or in close proximity). In one embodiment, said T cellsare primed T cells. In one embodiment, said tumor cells are in contactwith activated dendritic cells (or in close proximity).

In yet another embodiment, the present invention contemplates a systemcomprising first, second and third microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a bodyhaving a microchannel (optionally located centrally) therein; and an atleast partially porous membrane positioned within the microchannel (andoptionally along a plane), the membrane configured to separate themicrochannel to form a first microchannel and a second microchannel, themembrane comprising a top surface and a bottom surface, said i) topsurface comprises a first layer comprising living stromal cells, asecond layer positioned on top of said first layer and comprising livingepithelial cells, and living tumor cells in contact with said epithelialcells (or in close proximity), said ii) bottom surface comprising livingendothelial cells; said b) second microfluidic device comprising immunecells; and said c) third microfluidic device comprising anOrgan-on-Chip, (for example, a microfluidic device comprising cells ofan organ selected from the group consisting of cells of liver, kidney,lung, colon, intestine, brain, pancreas, skin (or other organ which canserve as a model for a distant metastasis). In one embodiment, saidmembrane is coated with at least one attachment molecule that supportsadhesion of a plurality of living cells. In one embodiment, said firstand second microchannels of said first microfluidic device comprisefluid. In one embodiment, said tumor cells are from a biopsy. In oneembodiment, said tumor cells are human tumor cells. In one embodiment,said tumor cells are also in contact with at least one type of immunecell (or in close proximity). In one embodiment, said tumor cells are incontact with lymphocytes (or in close proximity). In one embodiment,said tumor cells are in contact with T cells (or in close proximity). Inone embodiment, said T cells are primed T cells. In one embodiment, saidtumor cells are in contact with activated dendritic cells (or in closeproximity).

In yet another embodiment, the present invention contemplates a methodcomprising: 1) providing a) immune cells and b) a microfluidic devicecomprising a body having a microchannel (optionally located centrally)therein; and an at least partially porous membrane positioned within themicrochannel (and optionally along a plane), the membrane configured toseparate the microchannel to form a first microchannel and a secondmicrochannel, the membrane comprising a top surface and a bottomsurface, said i) top surface comprises a first layer comprising livingstromal cells, a second layer positioned on top of said first layer andcomprising living epithelial cells, and living tumor cells in contactwith said epithelial cells (or in close proximity), said ii) bottomsurface comprising living endothelial cells; and 2) introducing saidimmune cells into said microfluidic device under conditions such that atleast a portion of said immune cells contact said tumor cells. In oneembodiment, said membrane is coated with at least one attachmentmolecule that supports adhesion of a plurality of living cells. In oneembodiment, said first and second microchannels comprise fluid. In oneembodiment, said tumor cells are from a biopsy. In one embodiment, saidtumor cells are human tumor cells. In one embodiment, said immune cellsare introduced in step 2) in blood. In one embodiment, said immune cellscomprise lymphocytes and said tumor cells are in contact withlymphocytes (or in close proximity). In one embodiment, said lymphocytescomprise T cells. In one embodiment, said T cells are primed T cells. Inone embodiment, said immune cells comprise activated dendritic cells andsaid tumor cells are in contact with activated dendritic cells (or inclose proximity).

In yet another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device comprising a body having amicrochannel (optionally located centrally) therein; and an at leastpartially porous membrane positioned within the microchannel (andoptionally along a plane), the membrane configured to separate themicrochannel to form a first microchannel and a second microchannel, themembrane comprising a top surface and a bottom surface, said i) topsurface comprises a first layer comprising living stromal cells, asecond layer positioned on top of said first layer and comprising livingepithelial cells, and living tumor cells in contact with said epithelialcells (or in close proximity), said ii) bottom surface comprising livingendothelial cells; and 2) causing said immune cells in said firstmicrofluidic device to move into said second microfluidic device underconditions such that at least a portion of said immune cells contactsaid tumor cells. In one embodiment, said immune cells are exposed toone or more cytokines thereby causing said immune cells to move intosaid second microfluidic device. In one embodiment, fluidiccommunication is achieved through a conduit selected from the groupconsisting of a channel, a tube, or bridge, said conduit comprisingfluid. In one embodiment, said tumor cells are from a biopsy. In oneembodiment, said tumor cells are human tumor cells. In one embodiment,said immune cells of step 2) are in blood. In one embodiment, saidimmune cells comprise lymphocytes and said tumor cells are in contactwith lymphocytes (or in close proximity). In one embodiment, saidlymphocytes comprise T cells. In one embodiment, said T cells are primedT cells, said priming taking place in said first microfluidic device. Inone embodiment, said immune cells comprise activated dendritic cells andsaid tumor cells are in contact with activated dendritic cells (or inclose proximity).

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device and c) a third microfluidic device,said first microfluidic device comprising a body having a micro channel(optionally located centrally) therein; and an at least partially porousmembrane positioned within the microchannel and (optionally along aplane), the membrane configured to separate the microchannel to form afirst microchannel and a second microchannel, the membrane comprising atop surface and a bottom surface, said i) top surface comprises a firstlayer comprising living stromal cells, a second layer positioned on topof said first layer and comprising living epithelial cells, and livingtumor cells in contact with said epithelial cells (or in closeproximity), said ii) bottom surface comprising living endothelial cells;said third microfluidic device comprising cells of an organ selectedfrom the group consisting of cells of liver, kidney, lung, colon,intestine, brain; and 2) causing said immune cells in said firstmicrofluidic device to move into said second microfluidic device underconditions such that at least a portion of said immune cells contactsaid tumor cells. In one embodiment, said immune cells are exposed toone or more cytokines in said first microfluidic device thereby causingsaid immune cells to move into said second microfluidic device. In oneembodiment, fluidic communication is achieved through conduits, eachconduit selected from the group consisting of a channel, a tube, orbridge, said conduit comprising fluid. In one embodiment, said tumorcells are from a biopsy. In one embodiment, said tumor cells are humantumor cells. In one embodiment, said immune cells of step 2) are inblood. In one embodiment, said immune cells comprise lymphocytes andsaid tumor cells are in contact with lymphocytes (or in closeproximity). In one embodiment, said lymphocytes comprise T cells. In oneembodiment, said T cells are primed T cells, said priming taking placein said first microfluidic device. In one embodiment, said immune cellscomprise activated dendritic cells and said tumor cells are in contactwith activated dendritic cells (or in close proximity). In oneembodiment, said third microfluidic device comprises tumor cells incontact with said cells of an organ (or in close proximity). In oneembodiment, the method further comprises 3) causing said immune cells insaid first microfluidic device to move into said third microfluidicdevice under conditions such that at least a portion of said immunecells contact said tumor cells (or are in close proximity).

In still a further embodiment, the present invention contemplates amethod comprising: 1) providing a) living tumor cells and b) amicrofluidic device comprising a body having a microchannel (optionallylocated centrally) therein; and an at least partially porous membranepositioned within the microchannel (and optionally along a plane), themembrane configured to separate the microchannel to foam a firstmicrochannel and a second microchannel, the membrane comprising a topsurface and a bottom surface, said i) top surface comprises a firstlayer comprising living stromal cells, a second layer positioned on topof said first layer and comprising living epithelial cells, said ii)bottom surface comprising living endothelial cells; and 2) introducingsaid living tumor cells into said microfluidic device under conditionssuch that at least a portion of said living tumor cells contact withsaid epithelial cells. In one embodiment, the method further comprises3) incubating said living tumor cells in said microfluidic device, and4) determining whether said tumor cells invade said cell layers. In oneembodiment, said tumor cells are from a biopsy. In one embodiment, saidtumor cells are human tumor cells.

In yet another embodiment, the present invention contemplates amicrofluidic device comprising a gel and/or a membrane, said gel ormembrane comprising a top surface and a bottom surface, said a) topsurface comprises living epithelial cells, and living tumor cells incontact with said epithelial cells (or are in close proximity), said b)bottom surface comprising living endothelial cells. In one embodiment,said membrane is coated with at least one attachment molecule (e.g. ECMprotein) that supports adhesion of a plurality of living cells. In oneembodiment, the membrane separates first and second centralmicrochannels. In one embodiment, the microfluidic device comprises i) achamber, said chamber comprising a lumen and (optionally) projectionsinto the lumen, said lumen comprising ii) a gel matrix (optionallyanchored by said projections), said gel matrix positioned above iii) aporous membrane, said membrane positioned above iv) fluidic channels.Both the first and second central microchannels (mentioned earlier) andthe fluidic channels (mentioned above) may comprise fluid. In oneembodiment, said gel matrix is under a removable cover. In oneembodiment, at least a portion of said gel matrix is patterned. In oneembodiment, said gel matrix comprises collagen. In one embodiment, saidgel matrix is between 0.2 and 6 mm in thickness.

It is not intended that the present invention be limited by the natureor source of the tumor cells. In one embodiment, said tumor cells arefrom a biopsy. In one embodiment, said tumor cells are mammalian tumorcells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed Tcells). In one embodiment, said tumor cells are in contact withactivated dendritic cells (or in close proximity).

In yet another embodiment, the present invention contemplates a systemcomprising first and second microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a gel and/ora membrane, said gel or membrane comprising a top surface and a bottomsurface, said i) top surface comprises living epithelial cells, andliving tumor cells in contact with said epithelial cells (or in closeproximity), said ii) bottom surface comprising living endothelial cells;said b) second microfluidic device comprising immune cells. In oneembodiment, the first microfluidic device comprise a membrane, saidmembrane coated with at least one attachment molecule (e.g. ECM protein)that supports adhesion of a plurality of living cells. In oneembodiment, the membrane separates first and second centralmicrochannels. In one embodiment, the first microfluidic devicecomprises i) a chamber, said chamber comprising a lumen and (optionally)projections into the lumen, said lumen comprising ii) a gel matrix(optionally anchored by said projections), said gel matrix positionedabove iii) a porous membrane, said membrane positioned above iv) fluidicchannels. Both the first and second central micro channels (mentionedearlier) and the fluidic channels (mentioned above) may comprise fluid.In one embodiment, said gel matrix of said first microfluidic device isunder a removable cover. In one embodiment, at least a portion of saidgel matrix is patterned. In one embodiment, said gel matrix comprisescollagen. In one embodiment, said gel matrix is between 0.2 and 6 mm inthickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed T cells.In one embodiment, said tumor cells are in contact with activateddendritic cells (or in close proximity).

In still another embodiment, the present invention contemplates a systemcomprising first, second and third microfluidic devices in fluidiccommunication with each other, said a) first microfluidic devicecomprising a gel and/or a membrane, the gel or membrane comprising a topsurface and a bottom surface, said i) top surface comprising livingepithelial cells, and living tumor cells in contact with said epithelialcells (or in close proximity), said ii) bottom surface comprising livingendothelial cells; said b) second microfluidic device comprising immunecells; and said c) third microfluidic device comprising cells of anorgan selected from the group consisting of cells of liver, kidney,lung, colon, intestine, and brain. In one embodiment, the firstmicrofluidic device comprise a membrane, said membrane coated with atleast one attachment molecule (e.g. ECM protein) that supports adhesionof a plurality of living cells. In one embodiment, the membraneseparates first and second central microchannels. In one embodiment, thefirst microfluidic device comprises i) a chamber, said chambercomprising a lumen and (optionally) projections into the lumen, saidlumen comprising ii) a gel matrix (optionally anchored by saidprojections), said gel matrix positioned above iii) a porous membrane,said membrane positioned above iv) fluidic channels. Both the first andsecond central micro channels (mentioned earlier) and the fluidicchannels (mentioned above) may comprise fluid. In one embodiment, saidgel matrix of said first microfluidic device is under a removable cover.In one embodiment, at least a portion of said gel matrix is patterned.In one embodiment, said gel matrix comprises collagen. In oneembodiment, said gel matrix is between 0.2 and 6 mm in thickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed T cells.In one embodiment, said tumor cells are in contact with activateddendritic cells (or in close proximity).

In yet another embodiment, the present invention contemplates a methodcomprising: 1) providing a) immune cells and b) a microfluidic devicecomprising a gel and/or membrane, the gel or membrane comprising a topsurface and a bottom surface, said i) top surface comprising livingepithelial cells, and living tumor cells in contact with said epithelialcells (or in close proximity), said ii) bottom surface comprising livingendothelial cells; and 2) introducing said immune cells into saidmicrofluidic device under conditions such that at least a portion ofsaid immune cells contact said tumor cells. In one embodiment, themicrofluidic device comprises a membrane, said membrane coated with atleast one attachment molecule (e.g. ECM protein) that supports adhesionof a plurality of living cells. In one embodiment, the membraneseparates first and second central microchannels. In one embodiment, themicrofluidic device comprises i) a chamber, said chamber comprising alumen and (optionally) projections into the lumen, said lumen comprisingii) a gel matrix (optionally anchored by said projections), said gelmatrix positioned above iii) a porous membrane, said membrane positionedabove iv) fluidic channels. Both the first and second centralmicrochannels (mentioned earlier) and the fluidic channels (mentionedabove) may comprise fluid. In one embodiment, said gel matrix of saidfirst microfluidic device is under a removable cover. In one embodiment,at least a portion of said gel matrix is patterned. In one embodiment,said gel matrix comprises collagen. In one embodiment, said gel matrixis between 0.2 and 6 mm in thickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed T cells.In one embodiment, said tumor cells are in contact with activateddendritic cells (or in close proximity).

It is not intended that the present invention be limited by the nature,type or preparation of immune cells. In one embodiment, said immunecells are introduced in step 2) of the above-described method in blood.In another embodiment, said immune cells are introduced in step 2) inculture media. In one embodiment, the culture media flows as a flowrate.

In one embodiment, the above-described method has the further step of 3)introducing one or more agents (e.g. candidate drugs, known anti-cancerdrugs, known checkpoint inhibitors and candidate checkpoint inhibitors)into said microfluidic device. In one embodiment, the checkpointinhibitor is an antibody. In one embodiment, said antibody binds thePD-1 receptor on T cells. In one embodiment, said antibody binds thePD-L1 ligand on the tumor cells. In one embodiment, the method has thefurther step of 4) detecting (and/or measuring) the impact of the agenton the tumor cells, e.g. detecting tumor cell death by immune cells orby the agent.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device comprising a gel and/or membrane,the gel membrane comprising a top surface and a bottom surface, said i)top surface comprising living epithelial cells, and living tumor cellsin contact with said epithelial cells (or in close proximity), said ii)bottom surface comprising living endothelial cells; and 2) causing (atleast a portion of) said immune cells in said first microfluidic deviceto move into said second microfluidic device under conditions such thatat least a portion of said immune cells contact said tumor cells. It isnot intended that the method be limited to how the immune cells arecaused to move. In one embodiment, the immune cells are exposed toculture fluid at a flow rate. In one embodiment, said immune cells areexposed to one or more cytokines thereby causing said immune cells tomove into said second microfluidic device. In one embodiment, fluidiccommunication is achieved through a conduit selected from the groupconsisting of a channel, a tube, or bridge, said conduit comprisingfluid. In one embodiment, the second microfluidic device comprises amembrane, said membrane coated with at least one attachment molecule(e.g. ECM protein) that supports adhesion of a plurality of livingcells. In one embodiment, the membrane separates first and secondcentral microchannels. In one embodiment, the second microfluidic devicecomprises i) a chamber, said chamber comprising a lumen and (optionally)projections into the lumen, said lumen comprising ii) a gel matrix(optionally anchored by said projections), said gel matrix positionedabove iii) a porous membrane, said membrane positioned above iv) fluidicchannels. Both the first and second central microchannels (mentionedearlier) and the fluidic channels (mentioned above) may comprise fluid.In one embodiment, said gel matrix of said first microfluidic device isunder a removable cover. In one embodiment, at least a portion of saidgel matrix is patterned. In one embodiment, said gel matrix comprisescollagen. In one embodiment, said gel matrix is between 0.2 and 6 mm inthickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed Tcells). In one embodiment, said tumor cells are in contact withactivated dendritic cells (or in close proximity).

It is not intended that the present invention be limited by the nature,type or preparation of immune cells. In one embodiment, said immunecells are in blood. In another embodiment, said immune cells are inculture media. In one embodiment, the culture media flows as a flowrate.

In one embodiment, the above-described method has the further step of 3)introducing one or more agents (e.g. candidate drugs, known anti-cancerdrugs, known checkpoint inhibitors and candidate checkpoint inhibitors)into said microfluidic device. In one embodiment, the checkpointinhibitor is an antibody. In one embodiment, said antibody binds thePD-1 receptor on T cells. In one embodiment, said antibody binds thePD-L1 ligand on the tumor cells. In one embodiment, the method has thefurther step of 4) detecting (and/or measuring) the impact of the agenton the tumor cells, e.g. detecting tumor cell death by immune cells orby the agent.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device and c) a third microfluidic device,said second microfluidic device comprising a gel and/or membrane, thegel or membrane comprising a top surface and a bottom surface, said i)top surface comprising living epithelial cells, and living tumor cellsin contact with said epithelial cells (or in close proximity), said ii)bottom surface comprising living endothelial cells; said thirdmicrofluidic device comprising cells of an organ selected from the groupconsisting of cells of liver, kidney, lung, colon, intestine, brain; and2) causing said immune cells in said first microfluidic device to moveinto said second microfluidic device under conditions such that at leasta portion of said immune cells contact said tumor cells (or are in closeproximity). It is not intended that the method be limited to how theimmune cells are caused to move. In one embodiment, the immune cells areexposed to culture fluid at a flow rate. In one embodiment, said immunecells are exposed to one or more cytokines thereby causing said immunecells to move into said second microfluidic device. In one embodiment,fluidic communication is achieved through a conduit selected from thegroup consisting of a channel, a tube, or bridge, said conduitcomprising fluid. In one embodiment, the second microfluidic devicecomprises a membrane, said membrane coated with at least one attachmentmolecule (e.g. ECM protein) that supports adhesion of a plurality ofliving cells. In one embodiment, the membrane separates first and secondcentral microchannels. In one embodiment, the second microfluidic devicecomprises i) a chamber, said chamber comprising a lumen and (optionally)projections into the lumen, said lumen comprising ii) a gel matrix(optionally anchored by said projections), said gel matrix positionedabove iii) a porous membrane, said membrane positioned above iv) fluidicchannels. Both the first and second central microchannels (mentionedearlier) and the fluidic channels (mentioned above) may comprise fluid.In one embodiment, said gel matrix of said second microfluidic device isunder a removable cover. In one embodiment, at least a portion of saidgel matrix is patterned. In one embodiment, said gel matrix comprisescollagen. In one embodiment, said gel matrix is between 0.2 and 6 mm inthickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed Tcells). In one embodiment, said T cells are primed T cells, said primingtaking place in said first microfluidic device. In one embodiment, saidtumor cells are in contact with activated dendritic cells (or in closeproximity).

It is not intended that the present invention be limited by the nature,type or preparation of immune cells. In one embodiment, said immunecells are in blood. In another embodiment, said immune cells are inculture media. In one embodiment, the culture media flows as a flowrate.

In one embodiment, the above-described method has the further step of 3)causing said immune cells in said first microfluidic device to move intosaid third microfluidic device under conditions such that at least aportion of said immune cells contact said tumor cells. In oneembodiment, said third microfluidic device comprises tumor cells incontact with said cells of an organ (or in close proximity).

In one embodiment, the above-described method has the further step of 3)introducing one or more agents (e.g. candidate drugs, known anti-cancerdrugs, known checkpoint inhibitors and candidate checkpoint inhibitors)into said microfluidic device. In one embodiment, the checkpointinhibitor is an antibody. In one embodiment, said antibody binds thePD-1 receptor on T cells. In one embodiment, said antibody binds thePD-L1 ligand on the tumor cells. In one embodiment, the method has thefurther step of 4) detecting (and/or measuring) the impact of the agenton the tumor cells, e.g. detecting tumor cell death by immune cells orby the agent.

In yet another embodiment, the present invention contemplates a methodcomprising: 1) providing a) living tumor cells and b) a microfluidicdevice comprising a gel and/or membrane, the gel or membrane comprisinga top surface and a bottom surface, said i) top surface comprisingliving epithelial cells, said ii) bottom surface comprising livingendothelial cells; and 2) introducing said living tumor cells into saidmicrofluidic device under conditions such that at least a portion ofsaid living tumor cells contact with said epithelial cells. In oneembodiment, the method has the further steps of 3) incubating saidliving tumor cells in said microfluidic device, and 4) determiningwhether said tumor cells invade said cell layers. In one embodiment, themethod has the further steps of 3) introducing immune cells in saidmicrofluidic device, and 4) determining whether said immune cells causetumor cell death. In one embodiment, the above-described method has thefurther step of 3) introducing one or more agents (e.g. candidate drugs,known anti-cancer drugs, known checkpoint inhibitors and candidatecheckpoint inhibitors) into said microfluidic device. In one embodiment,the checkpoint inhibitor is an antibody. In one embodiment, saidantibody binds the PD-1 receptor on T cells. In one embodiment, saidantibody binds the PD-L1 ligand on the tumor cells. In one embodiment,the method has the further step of 4) detecting (and/or measuring) theimpact of the agent on the tumor cells, e.g. detecting tumor cell deathby immune cells or by the agent.

In one embodiment, the microfluidic device comprises a membrane, saidmembrane coated with at least one attachment molecule (e.g. ECM protein)that supports adhesion of a plurality of living cells. In oneembodiment, the membrane separates first and second centralmicrochannels. In one embodiment, the microfluidic device comprises i) achamber, said chamber comprising a lumen and (optionally) projectionsinto the lumen, said lumen comprising ii) a gel matrix (optionallyanchored by said projections), said gel matrix positioned above iii) aporous membrane, said membrane positioned above iv) fluidic channels.Both the first and second central microchannels (mentioned earlier) andthe fluidic channels (mentioned above) may comprise fluid. In oneembodiment, said gel matrix of said microfluidic device is under aremovable cover. In one embodiment, at least a portion of said gelmatrix is patterned. In one embodiment, said gel matrix comprisescollagen. In one embodiment, said gel matrix is between 0.2 and 6 mm inthickness.

Again, it is not intended that the present invention be limited by thenature or source of the tumor cells. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.Additional cells can also be included. For example, in one embodiment,the top surface further comprises a first layer comprising livingstromal cells, wherein said living epithelial cells comprise a secondlayer positioned on top of said first layer. As another example, in oneembodiment, said tumor cells are also in contact with at least one typeof immune cell (or in close proximity). A variety of immune cell typescan be used. In one embodiment, said tumor cells are in contact withlymphocytes (or in close proximity), including but not limited to tumorcells are in contact with T cells (which can be naïve or primed Tcells). In one embodiment, said tumor cells are in contact withactivated dendritic cells (or in close proximity).

In still another embodiment, the present invention contemplates amicrofluidic device comprising: a body having a first channel and afirst chamber; an at least partially porous membrane positioned at aninterface region between the first a channel and the first chamber, themembrane comprising a top surface and a bottom surface, said top surfacefacing the first chamber; living parenchymal cells disposed within thefirst chamber; and living tumor cells disposed within at least one ofthe first chamber or the first channel. In one embodiment, the firstchamber comprises a second channel. In one embodiment, the first chambercomprises an open region. In one embodiment, said living parenchymalcells comprise living epithelial cells. In one embodiment, the devicefurther comprises endothelial cells disposed within the first channel.In one embodiment, the living tumor cells are in contact with saidparenchymal cells. In one embodiment, the living tumor cells are incontact with said endothelial cells. In one embodiment, said membrane iscoated with at least one attachment molecule that supports adhesion of aplurality of living cells. In one embodiment, said first channelcomprises fluid. In one embodiment, said tumor cells are from a biopsy.In one embodiment, said tumor cells are human tumor cells. In oneembodiment, the device further comprises living stromal cells disposedwithin the first chamber. In one embodiment, said living stromal cellsare disposed in contact with the said top surface of the said membrane.In one embodiment, said living stromal cells are disposed within a gel.In one embodiment, said epithelial cells comprise a first layerpositioned on top of said living stromal cells. In one embodiment, saidstromal cells were derived from (or originated from) the site of atumor. In one embodiment, said stromal cells were derived from a siteaway from a tumor. In one embodiment, said stromal cells were derivedfrom a tumor-free sample. In one embodiment, the device furthercomprises at least one immune cell of at least one immune cell type. Inone embodiment, said at least one immune cell is in contact with saidtumor cells. In one embodiment, said at least one immune cell type islymphocytes. In one embodiment, said at least one immune cell type is Tcells. In one embodiment, said T cells are primed T cells. In oneembodiment, said at least one immune cell type is dendritic cells. Inone embodiment, said at least one immune cell was derived from (ororiginates from) within a tumor. In one embodiment, said at least oneimmune cell was derived from the proximity of a tumor. In oneembodiment, said at least one immune cell was derived away from theproximity of a tumor. In one embodiment, said at least one immune cellwas derived from a tumor-free sample. In one embodiment, said at leastone immune cell was derived from peripheral blood. In one embodiment,said tumor cells comprise at least one cell type corresponding to theorgan type represented (i.e. not metastasized) by at least some of thesaid parenchymal cells. In one embodiment, said tumor cells do notcorrespond (e.g. metastatic tumor) to the organ type represented by saidparenchymal cells.

In yet another embodiment, the present invention contemplates a systemcomprising first and second microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a bodyhaving a first channel and a first chamber; an at least partially porousmembrane positioned at an interface region between the first a channeland the first chamber, the membrane comprising a top surface and abottom surface, said top surface facing the first chamber; livingparenchymal cells disposed within the first chamber; and living tumorcells disposed within at least one of the first chamber or the firstchannel; said b) second microfluidic device comprising immune cells. Inone embodiment, said membrane is coated with at least one attachmentmolecule that supports adhesion of a plurality of living cells. In oneembodiment, said first and second central microchannels of said firstmicrofluidic device comprise fluid. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are human tumorcells. In one embodiment, the top surface further comprises a firstlayer comprising living stromal cells, wherein said living epithelialcells comprise a second layer positioned on top of said first layer. Inone embodiment, said tumor cells are also in contact with at least onetype of immune cell. In one embodiment, said tumor cells are in contactwith lymphocytes. In one embodiment, said tumor cells are in contactwith T cells. In one embodiment, said T cells are primed T cells. In oneembodiment, said tumor cells are in contact with activated dendriticcells. further comprising c) a third microfluidic device comprisingcells of an organ selected from the group consisting of cells of liver,kidney, lung, colon, intestine, and brain.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) an agent and b) a microfluidic devicecomprising: a body having a first channel and a first chamber; an atleast partially porous membrane positioned at an interface regionbetween the first channel and the first chamber, the membrane comprisinga top surface and a bottom surface, said top surface facing the firstchamber; living parenchymal cells disposed within the first chamber; andliving tumor cells disposed within at least one of the first chamber orthe first channel; and 2) introducing said agent into said microfluidicdevice. In one embodiment, the first chamber comprises a second channel.In one embodiment, the first chamber comprises an open region. In oneembodiment, said living parenchymal cells comprise living epithelialcells. In one embodiment, the method further comprises endothelial cellsdisposed within the first channel. In one embodiment, the living tumorcells are in contact with said parenchymal cells. In one embodiment, theliving tumor cells are in contact with said endothelial cells. In oneembodiment, said tumor cells are from a biopsy. In one embodiment, saidtumor cells are human tumor cells. In one embodiment, the method furthercomprises living stromal cells disposed within the first chamber. In oneembodiment, said living stromal cells are disposed in contact with thesaid top surface of said membrane. In one embodiment, said livingstromal cells are disposed within a gel. In one embodiment, saidepithelial cells comprise a first layer positioned on top of said livingstromal cells. In one embodiment, said stromal cells were derived fromthe site of a tumor. In one embodiment, said stromal cells were derivedfrom a site away from a tumor. In one embodiment, said stromal cellswere derived from a tumor-free sample. In one embodiment, the methodfurther comprises at least one immune cell of at least one immune celltype. In one embodiment, the at least one immune cell is in contact withsaid tumor cells. In one embodiment, said at least one immune cell typeis lymphocytes. In one embodiment, said at least one immune cell type isT cells. In one embodiment, said T cells are primed T cells. In oneembodiment, said at least one immune cell type is dendritic cells. Inone embodiment, said at least one immune cell was derived from within atumor. In one embodiment, said at least one immune cell was derived fromthe proximity of a tumor. In one embodiment, said at least one immunecell was derived away from the proximity of a tumor. In one embodiment,said at least one immune cell was derived from a tumor-free sample. Inone embodiment, said at least one immune cell was derived fromperipheral blood. In one embodiment, said tumor cells comprise at leastone cell type corresponding to the organ type represented by at leastsome of the said parenchymal cells. In one embodiment, said tumor cellsdo not correspond to the organ type represented by said parenchymalcells. In one embodiment, agent comprises a drug. In one embodiment,said agent comprises at least one of a toxin, a pollutant, a chemical, acosmetic. In one embodiment, said agent comprises a cell. In oneembodiment, said cell is an immune cell. In one embodiment, said immunecell is a T cell. In one embodiment, said T cell is a CAR-T cell. In oneembodiment, said agent comprises an immunotherapy agent. In oneembodiment, said agent comprises a chemotherapy agent. In oneembodiment, said agent comprises a checkpoint inhibitor. In oneembodiment, said agent comprises an antibody. In one embodiment, saidagent comprises at least one of anti-PD-1 or anti-PD-L1. In oneembodiment, the method further comprises 3) observing a response. In oneembodiment, said observing a response comprises detecting the death ofat least some of said tumor cells. In one embodiment, said observing aresponse comprises evaluating at least one of tumor size, tumor cellnumber, tumor metabolic activity, and tumor growth rate. In oneembodiment, said observing a response comprises evaluating at least oneof non-tumor cell death and non-tumor cell growth rate.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) immune cells and b) a microfluidic devicecomprising a body having a first channel and a first chamber, an atleast partially porous membrane positioned at an interface regionbetween the first a channel and the first chamber, the membranecomprising a top surface and a bottom surface, said top surface facingthe first chamber, living parenchymal cells disposed within the firstchamber; and living tumor cells disposed within at least one of thefirst chamber or the first channel; and 2) introducing said immune cellsinto said microfluidic device. In one embodiment, said membrane iscoated with at least one attachment molecule that supports adhesion of aplurality of living cells. In one embodiment, said first and secondcentral microchannels comprise fluid. In one embodiment, said tumorcells are from a biopsy. In one embodiment, said tumor cells are humantumor cells. In one embodiment, said immune cells are introduced in step2) in blood. In one embodiment, the top surface further comprises afirst layer comprising living stromal cells, wherein said livingepithelial cells comprise a second layer positioned on top of said firstlayer. In one embodiment, said immune cells comprise lymphocytes andsaid tumor cells are in contact with lymphocytes. In one embodiment,said lymphocytes comprise T cells. In one embodiment, said T cells areprimed T cells. In one embodiment, said immune cells comprise activateddendritic cells and said tumor cells are in contact with dendriticcells. In one embodiment, further comprising 3) introducing a checkpointinhibitor into said microfluidic device. In one embodiment, saidcheckpoint inhibitor is an antibody. In one embodiment, said antibodybinds the PD-1 receptor. In one embodiment, said antibody binds thePD-L1 ligand. In one embodiment, the method further comprises 4)detecting tumor cell death.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device comprising a body having a firstchannel and a first chamber, an at least partially porous membranepositioned at an interface region between the first a channel and thefirst chamber, the membrane comprising a top surface and a bottomsurface, said top surface facing the first chamber, living parenchymalcells disposed within the first chamber; and living tumor cells disposedwithin at least one of the first chamber or the first channel; and 2)causing said immune cells in said first microfluidic device to move intosaid second microfluidic device. In one embodiment, said immune cellsare exposed to one or more cytokines thereby causing said immune cellsto move into said second microfluidic device. In one embodiment, saidfluidic communication is achieved at least in part through a conduitselected from the group consisting of a channel, a tube, or bridge, saidconduit comprising fluid. In one embodiment, said fluidic communicationis achieved at least in part through discrete fluid transfers. In oneembodiment, at least some of said discrete fluidic transfer areperformed by at least one of a liquid-handling robot and autosampler. Inone embodiment, said tumor cells are from a biopsy. In one embodiment,said tumor cells are human tumor cells. In one embodiment, said immunecells of step 2) are in blood. In one embodiment, said immune cellscomprise lymphocytes. In one embodiment, said lymphocytes comprise Tcells. In one embodiment, the method further comprises 3) introducing acheckpoint inhibitor into said microfluidic device. In one embodiment,said checkpoint inhibitor is an antibody. In one embodiment, saidantibody binds the PD-1 receptor. In one embodiment, said antibody bindsthe PD-L1 ligand. In one embodiment, the method further comprises 3)detecting tumor cell death. In one embodiment, said T cells are primed Tcells, said priming taking place in said first microfluidic device. Inone embodiment, said immune cells comprise activated dendritic cells andsaid tumor cells are in contact with activated dendritic cells. In oneembodiment, the top surface further comprises a first layer comprisingliving stromal cells, wherein said living epithelial cells comprise asecond layer positioned on top of said first layer. In one embodiment,the method further comprises providing c) a third microfluidic device influidic communication with at least one of the first and secondmicrofluidic devices.

In still another embodiment, the present invention contemplates a methodcomprising: providing a) living tumor cells and b) a microfluidic devicecomprising a body having a first channel and a first chamber, an atleast partially porous membrane positioned at an interface regionbetween the first a channel and the first chamber, the membranecomprising a top surface and a bottom surface, said top surface facingthe first chamber, living parenchymal cells disposed within the firstchamber; and living tumor cells disposed within at least one of thefirst chamber or the first channel. In one embodiment, the methodfurther comprises 3) incubating said living tumor cells in saidmicrofluidic device, and 4) determining whether said tumor cells invadesaid cell layers. In one embodiment, said tumor cells are from a biopsy.In one embodiment, said tumor cells are human tumor cells. In oneembodiment, the method further comprises 3) introducing immune cellsinto said microfluidic device. In one embodiment, the method furthercomprises 4) evaluating at least one of tumor growth rate, tumor size,and tumor cell death, and non-tumor cell death. In one embodiment, themethod further comprises 3) introducing an agent into said microfluidicdevice. In one embodiment, the method further comprising 4) evaluatingat least one of tumor growth rate, tumor size, tumor cell death, andnon-tumor cell death. In one embodiment, said agent comprises at leastone of a drug, a toxin, a chemotherapy agent, an immunoncology agent, acheckpoint inhibitor, a chemical, and a cosmetic. In one embodiment,said agent is an antibody. In one embodiment, said antibody binds thePD-1 receptor. In one embodiment, said antibody binds the PD-L1 ligand.In some embodiments, the method involves additional cells on the device,such as stromal cells and/or endothelial cells. In one embodiment, thetop surface further comprises a first layer comprising living stromalcells, wherein said living epithelial cells comprise a second layerpositioned on top of said first layer. In one embodiment, the tumorcells may enter stroma. In one embodiment, the method further comprisesendothelial cell disposed in the device. In one embodiment, said tumorcells grow on said endothelial cells.

In still another embodiment, the present invention contemplates amicrofluidic device comprising: a body having a central microchanneltherein; and an at least partially porous membrane positioned within thecentral microchannel, the membrane configured to separate the centralmicrochannel to form a first central microchannel and a second centralmicrochannel, the membrane comprising a top surface and a bottomsurface, said a) top surface comprises living epithelial cells, andliving tumor cells in contact with said epithelial cells, said b) bottomsurface comprising living endothelial cells. In one embodiment, saidmembrane is coated with at least one attachment molecule that supportsadhesion of a plurality of living cells. In one embodiment, said firstand second central microchannels comprise fluid. In one embodiment, saidtumor cells are from a biopsy. In one embodiment, said tumor cells aremammalian tumor cells. In one embodiment, said tumor cells are humantumor cells. In one embodiment, the top surface further comprises afirst layer comprising living stromal cells, wherein said livingepithelial cells comprise a second layer positioned on top of said firstlayer. In one embodiment, said tumor cells are also in contact with atleast one type of immune cell. In one embodiment, said tumor cells arein contact with lymphocytes. In one embodiment, said tumor cells are incontact with T cells. In one embodiment, said T cells are primed Tcells. In one embodiment, said tumor cells are in contact with activateddendritic cells.

In still another embodiment, the present invention contemplates a systemcomprising first and second microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a bodyhaving a central microchannel therein; and an at least partially porousmembrane positioned within the central microchannel, the membraneconfigured to separate the central microchannel to form a first centralmicrochannel and a second central microchannel, the membrane comprisinga top surface and a bottom surface, said i) top surface comprises livingepithelial cells, and living tumor cells in contact with said epithelialcells, said ii) bottom surface comprising living endothelial cells; saidb) second microfluidic device comprising immune cells. In oneembodiment, said membrane is coated with at least one attachmentmolecule that supports adhesion of a plurality of living cells. In oneembodiment, said first and second central microchannels of said firstmicrofluidic device comprise fluid. In one embodiment, said tumor cellsare from a biopsy. In one embodiment, said tumor cells are mammaliantumor cells. In one embodiment, said tumor cells are human tumor cells.In one embodiment, the top surface further comprises a first layercomprising living stromal cells, wherein said living epithelial cellscomprise a second layer positioned on top of said first layer. In oneembodiment, said tumor cells are also in contact with at least one typeof immune cell. In one embodiment, said tumor cells are in contact withlymphocytes. In one embodiment, said tumor cells are in contact with Tcells. In one embodiment, said T cells are primed T cells. In oneembodiment, said tumor cells are in contact with activated dendriticcells.

In still another embodiment, the present invention contemplates a systemcomprising first, second and third microfluidic devices in fluidiccommunication, said a) first microfluidic device comprising a bodyhaving a central microchannel therein; and an at least partially porousmembrane positioned within the central microchannel, the membraneconfigured to separate the central microchannel to form a first centralmicrochannel and a second central microchannel, the membrane comprisinga top surface and a bottom surface, said i) top surface comprisingliving epithelial cells, and living tumor cells in contact with saidepithelial cells, said ii) bottom surface comprising living endothelialcells; said b) second microfluidic device comprising immune cells; andsaid c) third microfluidic device comprising cells of an organ selectedfrom the group consisting of cells of liver, kidney, lung, colon,intestine, skin and brain. In one embodiment, said membrane is coatedwith at least one attachment molecule that supports adhesion of aplurality of living cells. In one embodiment, said first and secondcentral microchannels of said first microfluidic device comprise fluid.In one embodiment, said tumor cells are from a biopsy. In oneembodiment, said tumor cells are mammalian tumor cells. In oneembodiment, said tumor cells are human tumor cells. the top surfacefurther comprises a first layer comprising living stromal cells, whereinsaid living epithelial cells comprise a second layer positioned on topof said first layer. In one embodiment, said tumor cells are also incontact with at least one type of immune cell. In one embodiment, saidtumor cells are in contact with lymphocytes. In one embodiment, saidtumor cells are in contact with T cells. In one embodiment, said T cellsare primed T cells. In one embodiment, said tumor cells are in contactwith activated dendritic cells.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) immune cells and b) a microfluidic devicecomprising a body having a central microchannel therein; and an at leastpartially porous membrane positioned within the central microchannel,the membrane configured to separate the central microchannel to form afirst central microchannel and a second central microchannel, themembrane comprising a top surface and a bottom surface, said i) topsurface comprising living epithelial cells, and living tumor cells incontact with said epithelial cells, said ii) bottom surface comprisingliving endothelial cells; and 2) introducing said immune cells into saidmicrofluidic device under conditions such that at least a portion ofsaid immune cells contact said tumor cells. In one embodiment, saidmembrane is coated with at least one attachment molecule that supportsadhesion of a plurality of living cells. In one embodiment, said firstand second central microchannels comprise fluid. In one embodiment, saidtumor cells are from a biopsy. In one embodiment, said tumor cells aremammalian tumor cells. In one embodiment, said tumor cells are humantumor cells. In one embodiment, said immune cells are introduced in step2) in blood. In one embodiment, the top surface further comprises afirst layer comprising living stromal cells, wherein said livingepithelial cells comprise a second layer positioned on top of said firstlayer. In one embodiment, said immune cells comprise lymphocytes andsaid tumor cells are in contact with lymphocytes. In one embodiment,said lymphocytes comprise T cells. In one embodiment, said T cells areprimed T cells. In one embodiment, said immune cells comprise activateddendritic cells and said tumor cells are in contact with activateddendritic cells. In one embodiment, the method further comprises 3)introducing a checkpoint inhibitor into said microfluidic device. In oneembodiment, said checkpoint inhibitor is an antibody. In one embodiment,said antibody binds the PD-1 receptor on T cells. In one embodiment,said antibody binds the PD-L1 ligand on the tumor cells. In oneembodiment, further comprising 4) detecting tumor cell death.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device comprising a body having a centralmicrochannel therein; and an at least partially porous membranepositioned within the central microchannel, the membrane configured toseparate the central microchannel to form a first central microchanneland a second central microchannel, the membrane comprising a top surfaceand a bottom surface, said i) top surface comprising living epithelialcells, and living tumor cells in contact with said epithelial cells,said ii) bottom surface comprising living endothelial cells; and 2)causing said immune cells in said first microfluidic device to move intosaid second microfluidic device under conditions such that at least aportion of said immune cells contact said tumor cells. In oneembodiment, said immune cells are exposed to one or more cytokinesthereby causing said immune cells to move into said second microfluidicdevice. In one embodiment, fluidic communication is achieved through aconduit selected from the group consisting of a channel, a tube, orbridge, said conduit comprising fluid. In one embodiment, said tumorcells are from a biopsy. In one embodiment, said tumor cells aremammalian tumor cells. In one embodiment, said tumor cells are humantumor cells. In one embodiment, said immune cells of step 2) are inblood. In one embodiment, said immune cells comprise lymphocytes andsaid tumor cells are in contact with lymphocytes. In one embodiment,said lymphocytes comprise T cells. In one embodiment, the method furthercomprises 3) introducing a checkpoint inhibitor into said microfluidicdevice. In one embodiment, said checkpoint inhibitor is an antibody. Inone embodiment, said antibody binds the PD-1 receptor on said T cells.In one embodiment, said antibody binds the PD-L1 ligand on the tumorcells. In one embodiment, the method further comprises 4) detectingtumor cell death. In one embodiment, said T cells are primed T cells,said priming taking place in said first microfluidic device. In oneembodiment, said immune cells comprise activated dendritic cells andsaid tumor cells are in contact with activated dendritic cells. In oneembodiment, the top surface further comprises a first layer comprisingliving stromal cells, wherein said living epithelial cells comprise asecond layer positioned on top of said first layer.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) a first microfluidic device comprisingimmune cells, said first microfluidic device in fluidic communicationwith b) a second microfluidic device and c) a third microfluidic device,said second microfluidic device comprising a body having a centralmicrochannel therein; and an at least partially porous membranepositioned within the central microchannel, the membrane configured toseparate the central microchannel to form a first central microchanneland a second central microchannel, the membrane comprising a top surfaceand a bottom surface, said i) top surface comprising living epithelialcells, and living tumor cells in contact with said epithelial cells,said ii) bottom surface comprising living endothelial cells; said thirdmicrofluidic device comprising cells of an organ selected from the groupconsisting of cells of liver, kidney, lung, colon, intestine, skin andbrain; and 2) causing said immune cells in said first microfluidicdevice to move into said second microfluidic device under conditionssuch that at least a portion of said immune cells contact said tumorcells. In one embodiment, said immune cells are exposed to one or morecytokines in said first microfluidic device thereby causing said immunecells to move into said second microfluidic device. In one embodiment,fluidic communication is achieved through conduits, each conduitselected from the group consisting of a channel, a tube, or bridge, saidconduit comprising fluid. In one embodiment, said tumor cells are from abiopsy. In one embodiment, said tumor cells are mammalian tumor cells.In one embodiment, said tumor cells are human tumor cells. In oneembodiment, said immune cells of step 2) are in blood. In oneembodiment, said immune cells comprise lymphocytes and said tumor cellsare in contact with lymphocytes. In one embodiment, said lymphocytescomprise T cells. In one embodiment, said T cells are primed T cells,said priming taking place in said first microfluidic device. In oneembodiment, said immune cells comprise activated dendritic cells andsaid tumor cells are in contact with activated dendritic cells. In oneembodiment, said third microfluidic device comprises tumor cells incontact with said cells of an organ. In one embodiment, the methodfurther comprises 3) causing said immune cells in said firstmicrofluidic device to move into said third microfluidic device underconditions such that at least a portion of said immune cells contactsaid tumor cells. In one embodiment, the top surface further comprises afirst layer comprising living stromal cells, wherein said livingepithelial cells comprise a second layer positioned on top of said firstlayer.

In yet another embodiment, the present invention contemplates a methodcomprising: 1) providing a) living tumor cells and b) a microfluidicdevice comprising a body having a central microchannel therein; and anat least partially porous membrane positioned within the centralmicrochannel, the membrane configured to separate the centralmicrochannel to form a first central micro channel and a second centralmicrochannel, the membrane comprising a top surface and a bottomsurface, said i) top surface comprising living epithelial cells, saidii) bottom surface comprising living endothelial cells; and 2)introducing said living tumor cells into said microfluidic device underconditions such that at least a portion of said living tumor cellscontact with said epithelial cells. In one embodiment, the methodfurther comprises 3) incubating said living tumor cells in saidmicrofluidic device, and 4) determining whether said tumor cells invadesaid cell layers. In one embodiment, said tumor cells are from a biopsy.In one embodiment, said tumor cells are mammalian tumor cells. In oneembodiment, said tumor cells are human tumor cells. In one embodiment,the method further comprises 3) introducing immune cells in saidmicrofluidic device, and 4) determining whether said immune cells causetumor cell death. In one embodiment, the method further comprises 5)introducing a checkpoint inhibitor in said microfluidic device, and 6)determining whether said checkpoint inhibitor causes tumor cell death.In one embodiment, said checkpoint inhibitor is an antibody. In oneembodiment, said antibody binds the PD-1 receptor on said T cells. Inone embodiment, said antibody binds the PD-L1 ligand on the tumor cells.In one embodiment, the top surface further comprises a first layercomprising living stromal cells, wherein said living epithelial cellscomprise a second layer positioned on top of said first layer.

In still another embodiment, the present invention contemplates amicrofluidic device lacking tumor cells, comprising: a body having a gelor an at least partially porous membrane, the gel or membrane comprisinga top surface and a bottom surface, said a) top surface comprises livingepithelial cells but lacking tumor cells, said b) bottom surfacecomprising living endothelial cells but lacking tumor cells, whereinsaid top surface, bottom surface or both surfaces of said membrane orgel comprise at least one attachment molecule that supports adhesion ofa plurality of living cells, wherein said at least one attachmentmolecule is derived from a tumor site of a patient. In one embodiment,said at least one attachment molecule is an extracellular matrixprotein. In one embodiment, the method further comprises immune cells.In one embodiment, said immune cells are derived from a tumor site of apatient.

In still another embodiment, the present invention contemplates a methodcomprising: 1) providing a) immune cells derived from a tumor site of apatient, said immune cells lacking contaminating tumor cells; and b) amicrofluidic device lacking tumor cells and comprising a gel or an atleast partially porous membrane, said membrane or gel comprising a topsurface and a bottom surface, said i) top surface comprising livingepithelial cells, said ii) bottom surface comprising living endothelialcells; and 2) introducing said immune cells into said microfluidicdevice under conditions such that at least a portion of said immunecells contact said epithelial cells, said endothelial cells or both. Inone embodiment, said membrane or gel comprises at least one attachmentmolecule that supports adhesion of a plurality of living cells. In oneembodiment, said at least one attachment molecule is derived from atumor site of a patient. In one embodiment, said at least one attachmentmolecule is an extracellular matrix protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows an exemplary schematic illustration describing embodimentsof hallmarks of cancer and tumor development.

FIG. 2: Shows an exemplary schematic illustration of translationalaspects and a micrograph of a cancer cell with attached immune cells.FIG. 2A: Shows an exemplary schematic illustration of embodiments ofhuman and mouse Cancer-on-Chip Provides a Mechanistic Insight and aBridge for Translation of treatments to humans. FIG. 2B: shows anexemplary colorized scanning electron micrograph of a tumor (purple)with attached immune cells (green).

FIG. 3: Shows an exemplary schematic illustration describing embodimentsof tumor growth. Tumors generate physical forces during growth andprogression: including but not limited to blood and lymphatic flow;mechanical stress (IHP, hypoxia); and decreased perfusion and hypoxiasuppress the immune response (pro-tumorigenic TAMs).

FIG. 4: Shows an exemplary schematic illustration of one embodiment forproviding a Tumor-On-Chip (Tumor-Chip) and one embodiment forincorporation of a tumor microenvironment. On the left, a schematicillustration shows one embodiment of a microfluidic Tumor-On-Chip (16),having two microfluidic channels (1), with an upper channel port (2) andlower channel port (3), with optionally used vacuum chambers (4). On theright, a schematic illustration shows one embodiment of a microfluidicTumor-On-Chip with four cell types, in the upper channel, tumor cellsand epithelial cells on top of a stromal cell layer separated by a chipmembrane from the lower channel with endothelial cells.

FIG. 5: Shows an exemplary schematic illustration of one embodiment forproviding Hypoxia-on-Chip. In the upper left chamber of FIG. 5A: Controlmedia gas concentration by bubbling a specific gas mixture through thechip into the receiving chamber in the upper right. FIG. 5B: The gasmixture changes the overall gas concentration in the system bycontrolling the gas concentration in media, ranging from 50-100% of thechosen gas, for one e.g. CO2.

FIG. 6: Shows an exemplary schematic illustration of several embodimentsfor providing Hypoxia-on-Chip which provides numerous options for OxygenGradients-on-Chip. FIG. 6A: Shows two examples of gradients that areformed by changing the input media gas concentration. Upper exampleshows media with low oxygen concentration limited to the upper part ofthe top channel while the lower example shows media with low oxygenconcentration in the lower part of the top channel and in the bottomchannel (see an exemplary gas key to the right). FIG. 6B: Show exemplaryschematic illustrations of several embodiments for providingHypoxia-on-Chip containing a tumor with three types of 02concentrations, from left to right, high concentration, lowconcentration below, in the lower channel and in the lower part of thetumor, and in the right schematic a low gas environment for the entiretumor.

FIG. 7: Shows exemplary micrographs of a forming tubular network aroundCollagen I Spheres. FIG. 7A: HuVEC-GFP forming tubular network aroundCollagen I Spheres. FIG. 7B: A higher power magnification of an area ofA (10×) showing Collagen I spheres (blue arrows) and red arrows pointingto network formation.

FIG. 8: Shows exemplary micrographs of a forming tubular network aroundCollagen I Spheres at high magnification. FIG. 8A: HuVEC-GFP formingtubular network around Collagen I Spheres. FIG. 8B: A higher powermagnification of the red outlined box area of A showing networkformation.

FIG. 9: Shows an exemplary schematic illustration of several embodimentsof emerging Hallmarks and enabling characteristics of Cancer.

FIG. 10: Shows an exemplary schematic illustration of one embodiment ofa cancer microenvironment, as one example, the lung cancermicroenvironment including but not limited to cells, cell receptors,signaling molecules, etc. found in a cancer microenvironment.

FIG. 11: Shows an exemplary schematic illustration of an open-topstretchable chip design as one embodiment of a chip used forCancer-On-Chip. FIG. 11A: Shows an exemplary schematic illustration of acircular format for a microfluidic channel, e.g. a bottom spiraledendothelial channel. FIG. 11B: Shows an exemplary schematic illustrationof parts of a microfluidic chip (16) including a lower circular formatfor a circular microfluidic channel, a membrane, and an upper part of achip. FIG. 11C: Shows an exemplary schematic illustration of anassembled microfluidic chip (16) showing a cross section of optionalvacuum channels in the upper part of the chip.

FIG. 12: Shows exemplary schematic illustrations of additional types ofchips that may be used for a Cancer-On-Chip, in part depending upon thetype of cancer/tumor. FIG. 12A: Shows an exemplary schematicillustration of a circular format (geometry) for a microfluidic chip.FIG. 12B: Shows exemplary photographs of a side view (upper) and topview (lower) circular chip. FIG. 12C: Shows an exemplary schematicillustration of a rectangular format (geometry) for a microfluidic chip.FIG. 12D: Shows exemplary photograph of a top view of a rectangularchip.

FIG. 13: Shows an exemplary schematic illustration and photographs ofone embodiment of a Skon-chip for culturing cancer cells as aCancer-On-Chip, e.g. metastatic melanoma. FIG. 13A: An exemplaryschematic illustration of one embodiment for Cancer-On-Chip. FIG. 13B:An exemplary photograph of Cancer-On-Chip shown in FIG. 13A. FIG. 13C:An exemplary photograph of Cancer-On-Chip shown in FIG. 13A attached tomicrofluidic connections.

FIG. 14: Shows exemplary fluorescent micrographs of cancer cells growingwithin a 3D environment, e.g. metastatic melanoma, immunostained forMiTF (microphthalmia-associated transcription factor) positive-cells(green) and nuclei shown in blue (DAPI staining). FIG. 14A: An exemplaryfluorescent micrograph showing cancer cells with MiTF positive-cells(green), nuclei (blue). In this view, cells express different levels ofMiTF where in some cell clusters many cells are expressing high levelsof MiTF (i.e. many light blue/green cells). FIG. 14B: An exemplaryfluorescent micrograph showing cancer cells where in this view, fewcells express MiTF (few light blue/green cells). FIG. 14C: An exemplaryfluorescent micrograph showing exemplary heterogeneity of tumor cells.Cancer cells with MiTF positive-cells (green), nuclei (blue) whereco-localized (combined channels) show light blue double stained nucleiof cancer cells.

FIG. 15: Shows exemplary micrographs of stained tissue sections showingmelanoma positive staining in cells cultured in cancer-on-chip. Amelanoma Cocktail stain, i.e. HMB-45/Mart-1/Tyrosinase, refers to amouse monoclonal antibody mixture (cocktail) used in routine clinicalpractice for the qualitative identification of human melanoma andmelanoma metastatic cells. For reference, HMB-45 antibody reacts with aneuraminidase sensitive oligosaccharide side chain of a glycoconjugate.Mart-1 refers to Melan-A: a melanoma antigen recognized by T cells.Tyrosinase refers to an oxidase enzyme (protein) which catalyzesreactions producing black/brown pigment within melanosomes. FIG. 15A:Shows an exemplary micrograph of melanoma cells staining with theMelanoma Cocktail (green) and nuclei (blue) at 11 days post seeding ofthe tumor in the Cancer-On-Chip. FIG. 15B: Shows an exemplary micrographof melanoma cells staining with a negative control using secondaryantibodies but not the primary antibodies in the Melanoma Cocktail.Nuclei are shown in blue.

FIG. 16: Shows exemplary micrographs of Melanoma Cocktail stained tissuesections showing melanoma positive cells cultured in cancer-on-chip.Melanoma Cocktail [HMB-45/Mart-1/Tyrosinase] is used in routine clinicalpractice for identification of Melanoma and Melanoma Metastases.Melanoma-on-Chip fixed, sectioned and stained with the MelanomaCocktail, 11 days post seeding. FIG. 16A: Shows an exemplary micrographof a stained section where cells stained positive for Melanoma markers(green). FIG. 16B: Shows an exemplary higher power magnified micrographof the area outlined in box 1 in FIG. 15A. FIG. 16C: Shows an exemplaryhigher power magnified micrograph of the area outlined in box 2 in FIG.15A.

FIG. 17: Shows an exemplary schematic illustration of a Cancer-Chip(Cancer-On-Chip) linked to a downstream (i.e. fluid receiving) LymphChip (Lymph Node-on-chip) that in turn has an upstream (i.e. fluidemitting) connection to the Cancer-Chip (i.e. circular fluidicflow/connection). Illustrations show examples of migratory immune cells.

FIG. 18: Shows an exemplary schematic illustration of numerousstimulatory and inhibitory factors in the cancer-immunity cycle. Chenand Mellman Immunity Rev. 2013.

FIG. 19: Shows exemplary schematic illustrations of embodiments forcomponents of a Microenvironment for Invasive Tumors. Douglas Hanahan,Robert A. Weinberg, 2011.

FIG. 20: Shows an exemplary schematic illustration of a Cancer-Chip(Cancer-On-Chip) linked to a Lymph Chip (Lymph Node-on-chip) as shown inFIG. 17, with at least one additional Organ-chip fluidically attached tothe Lymph Chip for providing a Metastasis-Chip (System). In oneembodiment, there is an incorporation of a vascular Component the LymphChip.

FIG. 21: Shows an exemplary schematic illustration of a Cancer-Chip(Cancer-On-Chip) fluidically linked to a Bone-Marrow Chip(Bone-Marrow-on-chip).

DEFINITIONS

Aspects described herein stem from, at least in part, design of devicesthat allow for a controlled and physiologically realistic co-culture oftumor cells with normal cells, whether together in one chamber of themicrofluidic device or separated by a membrane (or a combination ofboth). In one embodiment, the chambers of the microfluidic device arealigned (e.g., vertically) with each other with one or more membranesseparating tumor cells from other non-cancerous cells (“cancer chip”).The cancer chip devices have been developed and optimized based on thebasic design of an organ-on-a-chip as described in the U.S. Pat. No.8,647,861, and the International Patent App. No. PCT/US2014/071611, thecontents of each of which are incorporated herein by reference in theirentireties.

Tumor cells are contemplated to be placed in microfluidic devices orchips. As used herein, malignant neoplasia are referred to as “cancer”and characterized by tumor cells which typically will ultimatelymetastasize into distinct organs or tissues. Malignant neoplasia includesolid and hematological tumors. “Solid tumors” are exemplified by tumorsof the breast, bladder, bone, brain, central and peripheral nervoussystem, cervix, colon, endocrine glands (e.g. thyroid and adrenalcortex), esophagus, endometrium, germ cells, head and neck, kidney,liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas,prostate, rectum, renal, sarcoma, skin (e.g. melanoma), small intestine,stomach (or gastric cancer), soft tissue, testis, ureter, vagina andvulva. Malignant neoplasias include inherited cancers exemplified byRetinoblastoma and Wilms tumor. In addition, malignant neoplasia includeprimary tumors in said organs and corresponding secondary tumors indistant organs (“tumor metastases”). Hematological tumors areexemplified by aggressive and indolent forms of leukemia and lymphoma,namely non-Hodgkins disease, chronic and acute myeloid leukemia(CML/AML), acute lymphoblastic leukemia (ALL), Hodgkins disease,multiple myeloma and T-cell lymphoma. Also included are myelodysplasticsyndrome, plasma cell neoplasia, paraneoplastic syndromes, cancers ofunknown primary site as well as AIDS related malignancies (e.g. Kaposi'ssarcoma).

Agents are contemplated for testing on the cancer chip. A variety ofclasses of agents are contemplated, including but are not limited to (i)kinase inhibitors such as e.g. Glivec, ZD-1839/Iressa, Bay43-9006,SU11248 or OSI-774/Tarceva; (ii) proteasome inhibitors such as PS-341;(iii) histone deacetylase inhibitors like SAHA, PXD101, MS275, MGCD0103,Depsipeptide/FK228, NVP-LBH589, Valproic acid (VPA) and butyrates; (iv)heat shock protein inhibitors like 17-allylaminogeldanamycin (17-MG);(v) vascular targeting agents (VAT) and anti-angiogenic drugs like theVEGF antibody Avastin or the KDR tyrosine kinase inhibitorPTK787/ZK222584; (vi) monoclonal antibodies such as Herceptin orMabThera/Rituxan or C225/Erbitux as well as mutants and conjugates ofmonoclonal antibodies and antibody fragments; (vii) oligonucleotidebased therapeutics like G-3139/Genasense; (viii) protease inhibitors(ix) hormonal therapeutics such as anti-estrogens (e.g. Tamoxifen),anti-androgens (e.g. Flutamide or Casodex), LHRH analogs (e.g.Leuprolide, Goserelin or Triptorelin) and aromatase inhibitors. In oneembodiment, the PHSCN peptide is contemplated, in the form ofAc-PHSCN-NH₂ where all the amino acids are L-isomers or where one ormore amino acids are D-isomers as described in U.S. Pat. No. 8,940,701,hereby incorporated by reference.

In some embodiments, tumor cells are in contact with stromal cells (forexample, lamina propria-derived cells). As used herein, the term“stromal” refer to connective tissue cells including but not limited tomultipotent stromal cells (MSCs), e.g. Bone marrow derived mesenchymalstem cells, fibroblasts, myofibroblasts, mural cells (pericytes) of thevasculature, etc. Such cells may be found in or near sites ofinflammation, such as in or near the lamina propria in vivo, e.g.mucosa, submucosa, etc. Stromal cells are anything that isn'tparenchymal cells; lamina propria is a specific type of stroma. In someembodiments, stromal cells are contemplated for use in microfluidicdevices of the present inventions. In some embodiments, “stromal cells”are contemplated for use after isolation from lamina propria-derivedcells. In some embodiments, stromal cells are contemplated for usederived from regions that do not include lamina propria. In someembodiments, stromal cells are contemplated for use that are a mixtureof LP-derived and non-LP-derived cells, e.g. when biopsy tissue used forisolating cells includes both mucosa and submucosal cells. In someembodiments, stromal cells are isolated from healthy and diseasedindividuals, and/or from different sites within the same individual. Forexample, stromal cells may be from the site of a tumor vs. from ahealthy looking site.

In some embodiments, tumor cells are in contact with laminapropria-derived cells (or in close proximity). As used herein, the terms“lamina propria-derived cells” and “LP-derived cells” refers to cellsused in the context of specific tissues (e.g. mucosal tissues),including but not limited to stromal cells, fibroblasts, and residentimmune cells. In one embodiment, LP-derived cells are isolated fromspecific tissues (e.g. mucosal tissues). LP-derived cells are notlimited to mucosal tissues, as they may be isolated from tissuesextending into mucosal areas, for example, cells in stromal areas.LP-derived cells may be used directly after isolation or undergo cultureto expand cell numbers prior to use. LP-derived cells may undergoisolation techniques before or after culturing or freezing. In otherembodiments, LP-derived cells may be cryopreserved (frozen) prior touse.

As used herein, the phrases “linked,” “connected to,” “coupled to,” “incontact with” and “in communication with” refer to any form ofinteraction between two or more entities, including mechanical,electrical, magnetic, electromagnetic, fluidic, and thermal interaction.For example, in one embodiment, channels in a microfluidic device are influidic communication with cells and (optionally) a fluid reservoir. Twocomponents may be coupled to each other even though they are not indirect contact with each other. For example, two components may becoupled to each other through an intermediate component (e.g. tubing orother conduit).

“Channels” are pathways (whether straight, curved, single, multiple, ina network, etc.) through a medium (e.g., silicon, plastic, etc.) thatallow for movement of liquids and gasses. Channels thus can connectother components, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, liquid-intake ports and gas vents.

“Microchannels” are channels with dimensions less than 1 millimeter andgreater than 1 micron. Additionally, the term “microfluidic” as usedherein relates to components where moving fluid is constrained in ordirected through one or more channels wherein one or more dimensions are1 mm or smaller (microscale). Microfluidic channels may be larger thanmicroscale in one or more directions, though the channel(s) will be onthe microscale in at least one direction. In some instances the geometryof a microfluidic channel may be configured to control the fluid flowrate through the channel (e.g. increase channel height to reduce shear).Microfluidic channels can be formed of various geometries to facilitatea wide range of flow rates through the channels.

DESCRIPTION OF THE INVENTION

The invention generally relates to a microfluidic platforms or “chips”for testing and understanding cancer, and, more specifically, forunderstanding the factors that contribute to cancer invading tissues andcausing metastases. Tumor cells are grown on microfluidic devices withother non-cancerous tissues under conditions that simulate tumorinvasion. The interaction with immune cells can be tested to inhibitthis activity by linking a cancer chip to a lymph chip.

Hallmarks of cancer and tumor development: Cancer cells are abnormalcompared to healthy normal cells in the body. Cancer cells have numerouscharacteristics, e.g. uncontrolled cell cycles, which allow them tolive, replicate (grow), form tumors and/or spread throughout the body.Such growth typically results in severe illness and death unless thecancer is self-limiting (e.g. stops growing) and/or the immune system isable to control or irradicate these abnormal cells. See, schematicillustration of cancer cell characteristics in FIG. 1, Douglas Hanahan,Robert A. Weinberg, Cancer Cell 2011.

Several examples of conditions that affect cancer cell/tumor growth andcancer microenvironments include but are not limited to adjacent cells,e.g. a stromal component, an endothelial component, etc., and otherforces, such as physical and mechanical, e.g. oxygen availability, etc.

I. Cancer-On-Chip

As described herein, a Cancer-on-chip (Cancer-chip also Tumor-On-Chip orTumor-chip) provides a mechanistic insight and a bridge for translationof in vitro experiments to humans. In one embodiment, it provides ameans for translation of human in vitro experiments to humans. Inanother embodiment, it provides a means for translation of mouseexperiments to humans. See, schematic illustration of embodiments inFIG. 2.

In one embodiment, a Cancer-on-Chip is a human Cancer-on-Chip. However,it is not meant to limit the Cancer-on-Chip to human cells. In oneembodiment, a Cancer-on-Chip is a mouse Cancer-on-Chip. In oneembodiment, a Cancer-on-Chip is a rat Cancer-on-Chip. In one embodiment,a Cancer-on-Chip is a dog Cancer-on-Chip. Thus, a Cancer-on-Chip maycomprise any mammalian species. Further, in some embodiments, a mouseCancer-on-Chip is contemplated to be developed using cells fromtransgenic/humanized mouse models. In some embodiments, a comparison ofan animal Cancer-on-Chip model to human Cancer-on-Chip models are madefor determining applicability of certain animal to human translations.

A. Tumor Microenvironment: Cells, Tumors, Extracellular Matrix (ECM),Stroma and Immune Cells.

When a cancer cell forms a tumor, the tumor often provides amicroenvironment both surrounding, on or near the inside surface andfurther inside the tumor. These microenvironments may be very differentwhich is in part why these tumors are often difficult to study in vitro.Microenvironments are influenced by several components, including thecancer cells (which may have different types, e.g. nonmetastatic,premetastatic, metastatic, etc, and/or different levels of maturation,and other cell types that may be part of or surround the tumor, such asstromal cells, endothelial cells, etc., Further, the cells present makeextracellular matrix, including but not limited to collagen, varioustypes of hyaluronan, etc. Hyaluronic acid (HA; conjugate basehyaluronate), also called hyaluronan, refers to an anionic, nonsulfatedglycosaminoglycan. See, for one embodiment of exemplary components, FIG.3, Jain R K Cancer Cell 2014.

An artificial Biochemical Microenvironment of Cancer-On-chip providesgreater control of soluble factors required for cell function andsurvival through a recreation of the biochemical microenvironment withinthe Chips; Enables recreation of spatiotemporal gradients of solublefactors that allow cells to thrive in vivo; etc. The Fluidic nature ofthe Chips allows the epithelial and endothelial channels to remainfluidic independent (laminar flow), allowing different mechanical forcesprecisely controlled for each tissue, as well as, independentbiochemical signaling, and different use of cell culture medium or bloodcomponents in each channel. Continuous flowing cell culture media, bloodsubstitute or blood components bring in fresh nutrients, solublefactors, and dissolved gases, while washing away waste products; Canconnect different Organ-Chips allowing biochemical communication andsignaling between different Organ-Chips in a physiological manner; whereConventional cell culture and other 3D in vitro systems such asorganoids are static systems that fail to recapitulate in vivo dynamicsand appropriate biochemical microenvironments. Thus, Cancer-In-Chipsallow assessing developing tumors within the chip by several endpointsover the culture period: e.gs. growth and apoptosis rate of the cellswill be monitored over time; Effluent will be collected to assessconcentration of both dead and live cells; chips are amenable to realtime microscopy, as well as IHC and H&E staining; secreted biomarkerrelevant to the cell model will be assessed by the best available methodfor each factor tested. Biomarkers that can be monitored in the clinicalsetting will be prioritized; In addition, cells will be collected andprocessed for RNA seq and epigenetic profiling (also to confirm theresemblance to the tumor of origin); Transcriptomic and metabolomicsanalyses will be performed to further characterize the tumor grown onchip. This characterization will be conducted in basal state and will berepeated as additional cellular components are added in the chip andcomparisons between the different stages of development. An additionaladvantage of using Cancer-on-chip is that space constraints in the Chipprovide an additional potential advantage of the Chips, in that thespace limitations create a microenvironment similar to the confined,capsule-like structure, usually tumors grow in.

In one embodiment, a Cancer-on-chip provides a co-culture for tumorgrowth in a tumor microenvironment comprising multiple cellular, andrelated, components, for example, Tumor (cancer) cells, an epithelialcompartment, a stomal component, an endothelial compartment; see, forone embodiment, FIG. 4. Therefore, in one embodiment, a Cancer-on-chipprovides a co-culture for testing tumor invasion of the surroundingtissues, i.e. the epithelial, stroma, endothelial, etc., additionallyincluding exemplary environmental parameters that modulate cancer cellsand tumors include ECM and oxygen concentration, described below.

Methods of application of Cancer-on-chips are contemplated for studyingthe tumor biology, microenvironment, immune system interactions, testingof efficacy and safety of potential therapeutics, elucidation ofmechanisms of action, identification of biomarkers, system biologyapproaches, perturbations to microenvironment in cancer biology (e.g.presence of oncogenic factors in culture system). More specifically,pharmacology agents may be tested in these various contemplatedenvironments for identifying agents for blocking uncontrolled growth,metatasis and invasion of other cell layers, tissues, etc.

1. Cancer Cell Sources

In some embodiments, examples of cells for use on chips are obtainedfrom standard cell lines, primary tumor cells, primary tumor derivedmicrobiopsies, etc. Cancer cells may be human cells, mouse cells, orcancer cells of other mammalian species. As one exemplary method forseeding and using cancer cells, melanoma cells (e.g. well characterized)may be used in order to determine optical fluidic culture conditions.

In one embodiment, human tumor cell lines for use in Cancer chips arederived from tumors with immunogenic ability, such as the melanoma(A375) and lung cancer (A549) lines.

In one embodiment, cell line seeded Cancer-On-chip will parallelCancer-On-chip seeded with cultures of primary tumor cells. Primarytumor cells will be derived from freshly processed tumors/biopsysamples. Primary tumor cells may be derived, (e.g. isolated) fromspecific organs systems as well as patient specific cells, including butnot limited to healthy cells, cancer cells and tumor cells. In someembodiments, cancer cells isolated from an organ or tissue are not thesame type of cancer cells. For example, some lung cancer patients havesmall cell lung cancer while other patients have non-small cell lungcancer cells. Both types of cancer cells may find use in embodimentsdescribed herein for a lung Cancer-Cell-On-Chip. Moreover, tumor cellsemployed may not need to originate from the same organ type as othercells in the Organ-Chip. For example, pancreatic cancer cells may beused in a Liver-Chip to emulate a metastatic pancreatic cancer that hasmetastasized to the liver.

2. Endothelial Cells

In some embodiments, endothelial cells will be incorporated intoCancer-On-Chips for monitoring the interaction between tumor cells andendothelial cells, in particular for identifying factors leading toangiogenesis, and testing agents to inhibit such blood vessel formation,in addition to the formation of tumor vasculature, autophagy, etc.

In fact, incorporating endothelial cells in the lower (vascular) channelhas a positive impact on long-term tissue viability in general. Further,endothelialized chips have a closer resemblance to phenotypic andfunctional indices of the organ of origin of cells for seedingCancer-On-Chips.

Thus, in some embodiments, interactions with endothelial cells areprovided. For one example, microvascular endothelial lung cells would beco-cultured with lung cancer cells and tumors. In some embodiments,interactions with endothelial cells extend the longevity of the healthylung cells.

3. Healthy and Disease-Associated Materials

In some embodiments, the tumor microenvironment is explored by usingmaterials (ECM, cells etc.) found in or around the tumor (i.e.disease-associated materials)—with or without the tumor cellsthemselves—in the microfluidic devices described herein. In otherembodiments, materials (ECM, cells etc.) are used from healthy sources(healthy patients) or sites distant from the tumor site (cancerpatients)—with or without the tumor cells themselves—in the microfluidicdevices described herein. In some embodiments, disease-associatedmaterials are compared with healthy materials in the microfluidicdevices describe herein.

Some embodiments include using human cells and extracellar material,such as ECM, derived from each of these types of tissues. In particular,cells and/or at least one component isolated areas of tissue adjacent totumor areas and at sites of cancer cell or tumor growth, including butnot limited to types of cancer described herein.

As another example, lung is a frequent site of metastasis fromextrapulmonary neoplasms. Bacterial infection-inducedmetastasis-conducive environments in the lung and cigarettesmoke-induced inflammation are both associated with pulmonary metastasisfrom breast cancer. Further, such inflammation leads to the recruitmentof bone marrow-derived neutrophils enhancing metastatic outgrowth, e.g.Rayes, et al., “Inflammation promotes metastasis through neutrophilprotease-mediated degradation of Tsp-1.” Proc Natl Acad Sci USA.112(52): 16000-16005 (2015).

The following describes disease-associated materials contemplated formodeling and testing using a microfluidic device of the presentinventions.

4. Extracellular Matrix (ECM) in Cancer-On-Chips

The ECM configuration, and specific composition, effects establishmentof cancer cells and behavior of a developing tumor. Bhat and Bissell,“Of plasticity and specificity: dialectics of the micro- andmacro-environment and the organ phenotype.” Wiley Interdiscip Rev MembrTransp Signal. 3(2):147-163, 2014. Published online 2013. Thus, ECM foruse in Cancer-on-chips may be isolated from or near tumors in vivo. Onthe other hand, healthy ECM is contemplated for use as well, i.e. notassociated with cancer cell growth, etc., may be an isolated componentof ECM, may be a commercial source, etc.

In some embodiments, the central flexible, porous, membrane that dividesthe central channel of the chip into two compartments (epithelial andendothelial—vascular channels) is covered (coated) with an ECM. In someembodiments, the membrane is coated with extracellular matrix proteinsnative to the specific organ or disease state. In some embodiments, theECM promotes cell attachment and in vivo relevant organization of ECMand cell shape. In some embodiments, the cells under the in vivorelevant conditions recreated in the Chips also produce and modulatetheir own ECM, e.g. effecting progression of disease andmicroenvironment changes. In some embodiments, the ECM-cell interactionsmay effect cancer cell architecture, cell-cell communication, geneexpression and differentiation. In part because ECM interactions andresulting cell-cell communication in other in vitro systems such asorganoids lack in vivo relevance, in some embodiments, the ECMinteractions and resulting cell-cell communication in Cancer-on-chips iscontemplated to provide in vivo relevance.

In some embodiments, composition and stiffness of ECM is manipulated foridentifying negative or positive effects on cancer cell/tumor growth. Insome embodiments, determine ECM changes over co-culture time. In someembodiments, manipulate, e.g. overexpress/siRNA integrins or otherfactors.

As one specific example, the present invention in one embodimentcontemplates utilizing the basement membrane (BM) in breast tissue—withor without the actual cancer tissue on the microfluidic devicesdescribed herein. The basement membrane in breast tissue is aspecialized form of ECM linking epithelial and connective tissues, withadjacent stroma that traps an abundance of soluble factors constitutingthe microenvironment of the breast epithelium. More specifically,transmembrane integrins at the basal side of cells, having apical andbasal polarity, serve as anchorage points and receptors for BMcomponents. They trigger intracellular signaling and participate in theperception of the cells' microenvironment. They cooperate with growthfactor receptors to control essential cellular processes such assurvival, proliferation, and differentiation. Among the cell—BMcontacts, basal polarity is specifically determined by the interactionbetween laminin-332 and α6/β4 integrin dimers that form hemidesmosomes.Lateral cell—cell contacts are mediated by apical tight junctions,adherens junctions, and in some instances desmosomes. The tight sealgenerated by tight junctions prevents milk leakage in-between cellsduring lactation. The apical junctional complex formed by tight andadherens junctions also organizes the cytoskeleton and associatedsignaling pathways, which ultimately impinges on nuclear functions.Thus, the basoapical polarity axis permits unidirectional secretion ofmilk components in the lumen, as well as structured integration ofhormonal and mechanical signals exerted by the microenvironment. Vidi,et al., “Three-Dimensional Culture of Human Breast Epithelial Cells: TheHow and the Why.” Methods Mol Biol. 945:193-219, 2013.

5. Lamina Propria and Resident Immune Cells

Resident immune cells (B cells, T cells, dendritic cells, macrophages,and innate lymphoid cells) may be isolated from cancer patientsincluding from inflamed and non-inflamed regions of patient tissue. Inone embodiment, LP derived resident immune cells may be isolated fromsites of cancer/tumor cell growth. In one embodiment, LP derivedresident immune cells may be isolated away from sites of cancer/tumorcell growth. As one example, lamina propria-derived resident immunecells are used in a Cancer-On-Chip as described herein. In oneembodiment, tumor-associated immune cells are be isolated and includedin the Cancer-on-Chip. In one embodiment, tissue-specific residentimmune cells (e.g. Kupffer cells, Langerhans cells) are isolated andincluded in the Cancer-on-Chip.

In one embodiment, the present invention contemplates incorporatinglamina propria-derived cells (such as resident immune cells, e.g.leukocytes, (i.e. white blood cells), mononuclear cells, residentfibroblasts, etc.) in the chip embodiments described herein. Thus, inone embodiment, LPDCs are incorporated into an embodiment of theCancer-On-Chip. This can be done in a variety of combinations. In oneembodiment, the LPDCs, stromal cells, and/or resident immune cells aredeposited underneath epithelial cells and on top of an extracellularmatrix (ECM) composition coated membrane (e.g. with a gel overlay orsimply underneath the epithelial cells, i.e. without a gel overlay). Inone embodiment, the LPDCs, stromal cells, and/or resident immune cellsare further overlaid with a layer of ECM, i.e. ECM overlay, beforedepositing the epithelial layer. In one embodiment, however, the LPDCs,stromal cells, and/or resident immune cells are overlaid with an actualgel. In one embodiment, the LPDCs, stromal cells, and/or resident immunecells are deposited within a gel layer. The same or similar approachescan be used to incorporate other tissue-specific, stromal or residentcells (whether immune cells, fibroblasts, mixtures, etc.). In someembodiments, the LPDCs, stromal cells, and/or resident immune cells aredeposited within the endothelial channel, whether above, below co-mixed,or instead of with endothelial cells.

The lamina propria-derived cells or stromal cells can be used fordifferent degrees of purification or cell isolation: used wholesale,used with the cells isolated from ECM components, and isolated forspecific cell types. Thus, in one embodiment, a full milieu of celltypes was isolated and used in microfluidic devices described herein. Anexample of a full milieu of cell types used as a lamina propria-derivedcell population, include but are not limited to stromal cells,fibroblasts, and resident immune cells. Examples of stromal cellsinclude but are not limited to connective tissue cells, e.g.fibroblasts, myofibroblasts, etc., located in the mucosa, submucosa,etc. In fact, cells comprising LP-derived cells may not be limited tothe mucosa. In some embodiments, Examples of resident immune cellsincluding but are not limited to innate immune cells such as naturalkiller cells, γδ+ T cell receptor cells, adaptive immune cells, such asmononuclear cells, including monocytes, macrophages, basal cells,eosinophils, plasma cells, T cells, such as CD8+ CD4+, double positive,and dendritic cells, immature through mature, are found here. As anotherexample, purified/isolated LP-derived cell populations were used inmicrofluidic devices described herein. In some embodiments LP-derivedcells may be used directly after isolation. In some embodiments,LP-derived cells are expanded in cultures before adding to amicrofluidic chip of the present inventions.

Thus, in other embodiments, other types of purifications or isolationsare possible, including cells extracted from or isolated from laminapropria (as lamina propria derived cells, or LPDCs). In a preferredembodiment, resident immune cells are extracted and purified. In oneembodiment, lymphoid follicles are not included. In one embodiment,lymphoid follicles are included. In one embodiment, Payers patches arenot included. In one embodiment, Payers patches are included. Such thatthe presence of a lymphoid follicle or Payers patch in tissue used forisolation or extraction of cells may be determined by observation of thelamia propria tissue by optical microscopy prior to removal of cells. Inone embodiment, capillary endothelial cells are extracted and purified.

In one embodiment stromal tissue is used for isolation of stromal cells,LP derived cells, etc.

Other embodiments contemplated for mimicking disease is by manipulatingdifferentiation and/or activation stages of T cells. Thus, in yetanother embodiment, pre-differentiated T-cells are added to a chip ofthe present inventions. In one embodiment, the present inventioncontemplates the use of published protocols to differentiate naiveT-cells from peripheral blood mononuclear cells (PBMCs) isolated fromblood samples towards a Th9 T-helper cell fate comprising the use ofTGFb and IL4.

5. Multicellular Cytoarchitecture: Interactions with Stroma Cells/Stromaand Endothelial Cells/Vascularization.

In one embodiment, a Cancer-on-chip includes incorporation of stromalcomponent. Thus, in one embodiment, a Cancer-on-chip provides aco-culture for determining responses of stromal cells to tumor cells. Inone embodiment, a Cancer-on-chip provides a co-culture for determiningeffects of activated (tumor-derived) fibroblasts on tumor cell biology.In further embodiments, a Cancer-on-chip provides a co-culture forevaluating the activation of the stroma, interaction with tumor cells,and changes in the phenotype of tumor cells, following interaction withstroma, and effects on the vascular system such as changes inpermeability, neovascularization and metabolic function.

A stromal component may be incorporated into the Tumor-Chip by addingnormal fibroblasts, tumor fibroblasts, etc. In some embodiments, bothfibroblast types will be tested with the same tumor cells for comparingresults.

In particular, growth of tumor cells will be tested on top of stromalcells incorporated in a 3D-collagen gel or in separate channels wherethe stromal cells will be “housed” with the endothelial cells (asbelow), or with the stromal cells placed between the endothelial and theupper channel (tumor cells site). Characterization of growth parameterswill be characterized, including by Imaging studies with specificantibodies for confirming the identity, morphological characteristicsand properties obtained due to the co-culture in the Chip, e.g.identifying different cell types incorporated in the tumor-stroma Chips.Biochemical assays will assess secreting factors profiles and,transcriptomic analyses. The latter will be done to compare tumor cellstranscriptomics with or without stromal cells in the Chip.

In one embodiment, a Cancer-on-chip includes a vasculature component. Avascular component of our Chip design offers distinct advantages,including the ability to better recreate a tumor microenvironment and/ortissue-tissue interactions (e.g. epithelial and vascular, tumor andvascular). In addition, having the vascular component allows us to bringin circulating immune cells and the system further supports immune cellrecruitment (see Science 2010 Lung-on-Chip publication). Endothelialtissue added in the lower channel of the Chip can support flow from anindependent (and thus of different composition, if needed) mediumsource. In one embedment, the two fluidic channels in the Chip haveindependent flow and are controlled independently. In one embedment,endothelial cells may be used to determine neutrophil recruitment toareas of cancer cell/tumor growth. The inclusion of a vascular componentalso seems to improve the longevity and functional phenotype ofnon-tumor elements of Organ-Chips.

Cancer-on-Chips may have both a tissue and a vascular component in thetwo separate channels, i.e. stromal cells in the upper channel andendothelial cells in the lower channel. The vascular component of ourChip design offers distinct advantages, including the ability to betterrecreate a tumor microenvironment, tissue-tissue interactions (e.g.epithelial and vascular, tumor and vascular). In addition, having thevascular component allows bringing in circulating immune cells and thesystem further supports immune cell recruitment (see Science 2010Lung-on-Chip publication).

Conventional cell culture and other 3D in vitro systems such asorganoids lack appropriate tissue-tissue interface and appropriatemulticellular cytoarchitecture. Thus, Cancer-on-chips are designed forprovidng appropriate tissue-tissue interface and appropriatemulticellular cytoarchitecture. Thus, in one embodiment, aCancer-on-chip includes a Tissue-Tissue Interface and MulticellularCytoarchitecture. In some embodiments, through microengineeringtechniques we can direct the proper orientation of cells and theirinteractions with neighboring cells to recreate the in vivo situation.In some embodiments, Cancer-on-chips are designed to allow cells toreestablish essential tissue-tissue interfaces found in organs. In someembodiments, recreate multicellular architecture by adding more celltypes to increase complexity of the tissue within the Chips, e.g. oneembodiment of a Cancer-on-chip contains 4 different cell types(epithelial cells, liver sinusoidal endothelial cells, Kupffer cells(resident immune cells), and stellate cells (stromal cells).

In some embodiments, the present invention contemplates comparingregular fibroblast vs tumor fibroblasts, and adding myofibroblasts. Insome embodiments, the present invention contemplates determining theeffects of tumor on stromal differentiation (prognosis link). In someembodiments, modulate components to study effect on tumor growth.

In some embodiments, the present invention contemplates adding pericytesto Cancer Chip. In some embodiments, incorporate vascularization of the3D tumor. In some embodiments, the present invention contemplatesproviding changes in the microenvironment by inducing barrierperturbation (vascular leakage). In some embodiments, the presentinvention contemplates determining the effects of barrier perturbationon growth and metastasis of cancer cells. In some embodiments, thepresent invention contemplates determining the ability of tumor cells tomigrate into vasculature. In some embodiments, the present inventioncontemplates determining the effects of +/− endothelial cells, allowinga mechanistic understanding of the vascular component.

B. Tumor Microenvironment-Tumor Growth: Physical Forces.

Tumors may generate physical forces during growth and progression, fornonlimiting examples, blood and lymphatic flow, mechanical stress (e.g.intermittent hydrostatic pressure (IHP), hypoxia, etc.). The term“hypoxia” as used herein refers to a deficiency in oxygen. Thus, in someembodiments, elements of a Hypoxia-on-Chip are used in combination witha Tumor chip.

In some embodiments, label cells to follow growth and migration throughimaging. In some embodiments, change oxygen levels, e.g. induce hypoxiaconditions. In some embodiments, change mechanical pressures. Seesections below describing these conditions in more detail.

1. Oxygen Concentration and Hypoxic Environment: Hypoxia-On-Chip

Tumors thrive in the hypoxic environment created as they expand in size.One main area of focus will be to recreate relevant hypoxic conditionswithin Cancer Chips and engineer a control that allows dynamicmodulation of the oxygen concentration of the tumor microenvironment, inpart, to study the impact of oxygen concentration on tumor growth withinthe Chip. Tumor development will be tested in normoxic and hypoxicconditions that will allow modulation of oxygen concentrations undercontrolled and regulated conditions.

In some embodiments, change the overall gas concentration in the systemby controlling the gas concentration in media. In some embodiments,generate oxygen gradients on chip from cellular consumption of oxygen.In some embodiments, change the magnitude of the oxygen gradient byvarying input oxygen concentration. In some embodiments, change theslope of the oxygen gradient by varying the input flow rate. In someembodiments, set oxygen concentration (mol/m^3) due to hepatocyte oxygenconsumption in Tall Channel at a flow of 250 uL/hr.

Thus, in some embodiments, changing oxygen levels induces hypoxia foreffecting tumor growth, invasion and migration. In some embodiments,reduced oxygen or hypoxia at or near the tumor may be generated byperfusion with fluid with reduced oxygen (or dissolved oxygen)concentration. In some embodiments, such reduced oxygen or hypoxia maybe attained by disposing the Cancer-on-Chip or a portion thereof into areduced oxygen environment (e.g. an hypoxic chamber).

2. Mechanical Forces

Mechanotransduction of mechanical forces in cells are determinants ofcellular function, cell signaling, and gene expression and implicated indevelopmental biology. For examples, Cells in vivo experience mechanicalforces via various mechanisms e.g. Expansion of lungs during breathing;Flow of air over cilia of epithelial cells in the airway; Flow of bloodcreating shear stress forces on vascular endothelium that can alsoimpact epithelial cell function; Peristalsis in the intestine, etc.Further, mechanotransduction effects cell function and diseasedevelopment including inflammation and immune response. Mechanicalforces and mechanotransduction are not considered in other in vitromodels including organoid models.

Thus, embodiments of Cancer chips may include mechanical forces, such asflow rates that generate physiological shear stress forces, orstretching of the chip.

a. Hydrostatic Pressure: Interstitial Fluid Pressure (IFP)

Solid tumors may have a raised interstitial fluid pressure (IFP) due, inpart, to high vessel permeability, low lymphatic drainage, poorperfusion, and high cell density around the blood vessels.

Decreased perfusion and hypoxia suppress the immune response, andencourage pro-tumorigenic tumor-associated macrophages (TAMs)).

Some embodiments are contemplated for using human and mouse systems, foruse separately and together for humanized systems.

In some embodiments, the effect of IFP is emulated by applying a fluidpressure the Cancer-on-Chip. For example, pressure may be appliedthrough one of more fluidic channels that the Cancer-on-Chip comprises.Such pressure can be generated by means including by not limited tohydrostatic head, piston pressure, pneumatic actuation (e.g. ofliquids), and a combination thereof. In other embodiments, IFP isemulated by the direct application of mechanical force to the site ofinterest (e.g. the tumor and/or its environment). For example, this canbe accomplished through the direct action of a piston. In someembodiments, the IFP is modulated through the duration of theexperiment. For example, it may be increased over time to mimic agrowing tumor, or it may be varied cyclically.

b. Additional Mechanical Forces

In some embodiments, mechanical forces may include: shear stress,compressive forces, tensional forces, cell traction forces, cellpre-stress, etc.

II. Cancer-On-Chip with Immune Cells.

Immune cells are key mediators of inflammation and play important rolesin diseases states such as cancer. Fluidic nature of the system allowsimmune cells to be introduced into the system in a dynamic manner, e.g.flow neutrophils or macrophages into the Chip, flow in immune cells fromother organ systems, and introduce resident immune cells into the tissuewithin the Chip. Ability to study complex interactions between bloodcomponents. Conventional cell culture and other 3D in vitro systems suchas organoids lack fluidic/dynamic nature and ability to flow in immunecells from other organ systems or to study hemodynarnics in vitro.

Thus, in some embodiments, immune cells are incorporated into theTumor-Chip by flowing immune cells such through the Chip to successfullyrecapitulate this aspect of the tumor-immune cell interactions. Theinteraction of immune cells with tumor cells and endothelial cells willbe assessed in real-time, e.g. real-time evaluation of chemotacticactivity, including diapedesis to the epithelial layer. The goal on thisstep is to confirm that we can observe in the Chip the demonstrated,positive and negative interactions, of tumor cells with specific immunetypes, such as the CD8+ T cells, NK cells, Treg and myeloid suppressorcells. The major challenge here will be procurement of the cells andquality of the immune cells as well as obtaining matched cells from samedonors for the different tissue within the chip. Exemplary steps fordeveloping a Tumor-Chip include, but are not limited to, design,engineer, optimize, and characterize; mouse tissue used in chips (e.g.proof of concept: poc) and human chips, including but not limited toadding or developing myeloid suppressors.

A. Cancer-On-Chip with Blood Immune Cells and Blood Components

Given the role of recruitment of circulating immune cell andinflammatory responses in disease etiology, it is desired that thesecomponents be integrated into engineered in vitro disease models, anachievement that is now possible using microengineered and fluidic-basedCancer-on-Chip systems.

The interaction between cancer cells/tumors and circulating peripheralwhite blood cells, and other blood components, influences cancer celland tumor viability, along with metastasis.

Thus, in one embodiment, incorporate immune cells into the Tumor-Chip byflowing immune cells such as PBMCS through the Chip to successfullyrecapitulate this aspect of the tumor-immune cell interactions.

The interaction of immune cells with tumor cells and endothelial cellswill be assessed in real-time. The goal on this step is to confirm thatwe can observe in the Chip the demonstrated, positive and negativeinteractions, of tumor cells with specific immune types, such as theCD8+ T cells, NK (Natural Killer) cells, Treg and myeloid suppressorcells. The major challenge here will be procurement of the cells andquality of the immune cells as well as obtaining matched cells from samedonors for the different tissue within the chip.

In one embodiment, observation of in vivo relevant dynamic interactionsbetween tumor cells and cells is contemplated to determine the specificimpact of the endothelium on this interaction over time. Therefore, thisartificial system will allow one to address questions such as the orderof events in the interaction of tumor and endothelial cells as well asgain mechanistic understanding. The in vivo models are not alwayshelpful in elucidating such mechanisms as they provide a whole animalview, rather than cellular resolution view. In one embodiment, acomparison of these microfluidic chip based in vitro studies to in vivostudies is contemplated.

1. Immune Cells

In further embodiments, an immune component is incorporated. Supply ofspecific human immune cells may be from peripheral blood cells of apatient, healthy, precancerous or diagnosed with cancer, with or withoutundergoing treatment. In one embodiment, in vitro studies incorporatingimmune cells are engineered to be translated into in vivo studies.

Immune interactions may be local (by adding cells to chips). Thus, inone embodiment, at least one type of immune cell is added to aCancer-In-Chip. In one embodiment, incorporation of monocytes in theCancer-on-Chip is contemplated, in addition to different cell types,e.g. epithelial, endothelial, stromal and resident immune cells, inaddition to cancer cells or tumors.

2. Tumor-Infiltrating Lymphocytes (TILs)

In yet another embodiment, Tumor-infiltrating lymphocytes (TILs) areadded to a Cancer-In-Chip. In some embodiments, TILs may be isolatedfrom tumors for adding to chips. In some embodiments, TILs may bederived from infiltrating immune cells added to chips, as describedherein.

In one embodiment, compartmentalize areas containing at least one typeof immune cell is provided within the chip. Thus, in one embodiment, anisolated lymph node, e.g. isolated from a subject, e.g. healthy,patient, cadaver, etc., may be added to a Cancer-In-Chip. Thus, in oneembodiment, T cells are introduced into Cancer chips. In one embodiment,antigen-presenting cells are introduced into Cancer chips.

Thus, in one embodiment, gradually build the chip's complexity in partby adding different types of immune cells.

Selection of T cells and myeloid suppressor cells for adding to thesecultures, address potential adverse immunological reactions due to lackof histocompatibility. Thus in some embodiments, immune cells areautologous to cancer cells. In some embodiments, immune cells areengineered to reduce immune reactions due to MHC mismatch. In someembodiments, cancer cells are naturally or engineered to reduce MHCmismatch immune stimulation.

III. Additional Chips for Linking with Cancer-On-Chip

The ability to link multiple Organs-on-Chips (via exposure of effluentor direct linking of multiple tissues) would also enable the study ofdynamic interactions between different organs systems that are known tobe essential in tumor biology. Therefore, in combination with aTumor-on-Chip, immune system chips may be fluidically linked forimitating an immune system, including the emulation of Lymph node orThymus-on-Chip, and the Bone Marrow. An advantage is that thearchitecture of these organs is well characterized to guide theengineering to the incorporation of the essential components in anorderly manner, i.e. one at a time incorporation into the Cancer chipsystem. The caveat is that they are constitutively active organs, with arange of dynamic regulatory functions, and their functions are finelymodulated by a number of stimuli.

Further, embodiments are contemplated to gradually build the system'scomplexity, in part, by linking different types of chips comprisingspecific cell types, such as epithelial cells, immune system cells, etc.for modeling specific cell-cell interactions and/or interactions ofcancer cells/tumor with dynamic systems, such as Lymph node-chips, Bonemarrow-chips, etc. Thus, in yet other embodiments, systems on separatechips are linked with Cancer-on-Chip, e.g. stromal-chip,endothelial-chip, epithelial-chip, immune-system chip, etc.

In one embodiment, an immune cell may be added to a Cancer-On-chip by asystemic simulation (i.e. linking chips, e.g. Lymph node chip) togetherwith a Cancer-In-Chip. Thus, in one embodiment, a Cancer-on-chipprovides a co-culture system for tumor interactions with the lymphaticsystem.

Further, embodiments are contemplated to gradually build the system'scomplexity, in part, by linking different types of chips comprisingimmune system cells. In yet further embodiments, immune system chips,e.g. Lymph Node-Chip, Bone-Marrow-Chip, Thymus-Chip, DC-Chip, etc. arelinked with Cancer-on-Chip. Therefore we propose to develop theTumor-on-Chip with an immune system simulation, including the emulationof Lymph node Thymus-on-Chip, and Bone Marrow-on-Chip, described below.

The ability to link multiple Organs-on-Chips (via exposure of downstreameffluent or by direct circular linking of multiple tissues) would alsoenable the study of dynamic interactions between different organssystems and Cancer-on-Chip that are known to be components in tumorbiology.

The interaction between tumor and lymph nodes or bone marrow will beachieved by linking the Tumor-Chip with an Immune System-Chip, such aslymph node or bone marrow. Thus communication will be established viaengineered gradients of media containing chemokines and/or other tumorchemotactic factors that will be recirculating to reveal successfulfunctional interaction between the two, to be assessed by relevantmarkers and cell changes characterization. We anticipate that successfullinkage should be able to resemble lymphatic drainage of the tumor to atissue, and then expand the linkage to include additional, distantorgans, normal stromal, normal endothelial cells, etc.

After linking, immune chips, additional studies are contemplated toexplore functionality for a number of processes requiring immune systemand tumor interactions such as: Tumor-associated inflammation; effect ofstroma and/or tumor cells in mobilization of immune cells by therespective organs and the subsequent efficacy of immunotherapy (in aneffort to simulate early and late effects in the course of tumorexpansion) to control the metastatic capacity of the original tumor,etc.

A. Lymph Node-Chip

In some embodiments, a lymph node-chip is linked to a Tumor-Chip.Exemplary steps for developing a lymph node-chip include, but are notlimited to, design, engineer, optimize, and characterize; mouse tissueused in chips (e.g. proof of concept: poc) and human chips. Thus, in oneembodiment, a system is provided for tumor-immune system interactions bylinking a Cancer-on-Chip to a Lymph Node-Chip. Such a chip iscontemplated to have Lymph Node-relevant architecture, in- and out-flow,etc., for simulation of draining lymph node function.

A Lymph Node-Chip is designed to recapitulate the entry of antigenpresenting cells (APCs) in the lymph node, the contact between APCs andT cells residing in the lymph node and the traveling of T cells to(and/or from) the tumor. APCs may need pre-activation (in addition toactivation via tumor cells contact). In one embodiment, a gradient ofCCL19, CCL21 is created inside the lymph node chip to recapitulate thein vivo microenvironment.

As one example of an embodiment to provide effects of CTLs on cancercells, cytotoxic cells will be dyed with a cell dye to be distinct andfollowed by microscopy. In one embedment, a CTL will be exposed to theprimed DC and then will be circulated in the system through a cancerchips's vascular system. CD8+ T cells may be prohibited from enteringthe tumor, e.g. stacked in the stroma, a response that could be modifiedby specific immunotherapies. Such that, such a chip might recapitulatedtumor “non immune-permissive” environment, in part, to proceed withtesting of therapeutic approaches. Our goal is that antigen-presentingcells (dendritic) perfused in the tumor, will be interacting with Tcells in the lymph node-on-chip. Next, T cells “educated” by DCs throughan engineered closed circuit will be driven to the tumor, viadevelopment of chemokine gradients or similar approaches, to assesstheir interaction (or not) with the tumor. As expected by the in vivoconditions, the CD8+ T cells should be prohibited from entering thetumor and should be stacked in the stroma, or if entered in the tumorwill show no cytotoxic activity (exhausted T cells), a response thatcould be modified by specific checkpoint inhibitors. Recruitment of theeducated T cells to the tumor site will be driven by engineered fluidicpressure differences, and if needed by developed chemokine gradients.

In one embodiment, migration/attraction of activated T cells from thelymph node chip back to the tumor chip is observed. In one embodiment,flow is used for migration. In one embodiment, antigen (Ag) presentationto T cells suffices to attract immune cells to the tumor site. In oneembodiment, a tumor chip gradient of chemoattractants (e.g. chemokine)is established when Ag presentation to T cells does not suffice toattract immune cells to the tumor site.

B. Lymph Node-Chip and Circulation/Metastasis.

The interaction between tumor and lymph nodes or bone marrow will beachieved by linking the Tumor-Chip with an Immune System-Chip, such aslymph node or bone marrow. Thus communication will be established viaengineered gradients of media containing chemokines and/or other tumorchemotactic factors that will be recirculating to reveal successfulfunctional interaction between the two, to be assessed by relevantmarkers and cell changes characterization. We anticipate that successfullinkage should be able to resemble lymphatic drainage of the tumor to atissue, and it is possible we will then expand the linkage to includeadditional, distant organs or just normal stromal or endothelial cells.

As one example, lung cancer is modeled in relation to draining lymphnodes. Thus, in one embodiment, a normal Lung-Chip is linked to a LymphNode-Chip interconnected with a Melanoma Chip to provide a system formelanoma metastasis to lung tissue.

C. Bone Marrow-Chip

A Bone Marrow-Chip is provided as a source for immune cells that will beattracted by—and recruited to—the Tumor-Chip through fluidiccommunications. Thus, immune cells are incorporated into the Tumor-Chipby flowing immune cells originated from the Bone Marrow-Chip torecapitulate this aspect of the tumor biology. In one embodiment, a BoneMarrow-Chip is a microengineered model that replicates native niche andkey immunological function of human bone marrow in vivo. In oneembodiment, a Bone Marrow-Chip is a mouse Bone Marrow-Chip. In oneembodiment, a Bone Marrow-Chip is a human Bone Marrow-Chip.

In one embodiment, a Bone Marrow-Chip in fluidic communication with aCancer chip recapitulates tumor-mediated signals to the bone marrow totrigger proliferation of relevant progenitors and induce mobilization ofmyeloid cells that populate the tumor itself. IN one embodiment, thisplatform enables maintenance of the physiological bone marrowmicroenvironment and production of genetically altered neutrophils invitro from retroviral-transduced hematopoietic stem cells, an extremelydifficult task. Furthermore, this in vitro system makes it possible toretain the microvasculature within the marrow and to simulatemobilization and recruitment of neutrophils using chemokines andcolony-stimulating factors.

A prototype system will be constructed by incorporating human bonemarrow obtained surgically from thoracectomy into a perfusable Chip thatcontains a vascularized three-dimensional tissue culture scaffold. Thedesign of this model will enable spontaneous anastomosis of themicrovasculature in the marrow with a network of microengineered bloodvessels, making it possible to generate and precisely control vascularperfusion of the bone marrow. Once this culture is established, humanhematopoietic stem cells will be introduced into the engineered tissueand induced to differentiate into the myeloid lineage. Functionalvalidation of this model will be initially achieved by measuringmobilization of neutrophils in response to colony-stimulating factorssuch as G-CSF or CXC chemokines such as IL-8. The model will be appliedto the Cancer-Chip using effluent form the tumor chip to mobilizemyeloid suppressor cells and monocytes and measure their ability to getincorporated within the Tumor-on-Chip. Actual linking between thedifferent chips will be done as outlined herein.

D. Linking Immune System-On-Chips: Draining Lymph Node-On-Chip and/orBone Marrow-On-Chip (and or Other Immune-Chips) with Cancer-On-Chip.

In one embodiment, a Draining Lymph Node-Chip and/or Bone Marrow-Chip isfluidically linked with a Cancer-On-Chip, in part, for demonstratingrecruitment of bone marrow cells by the Cancer-On-Chip. In oneembodiment, a rodent Bone-marrow-on-Chip is used in combination with ahuman cancer chip. In one embodiment, a rodent Bone-marrow-on-Chip willfacilitate translation to human Bone Marrow-Chip model.

In one embodiment, a Draining Lymph Node-Chip and/or Bone Marrow-Chip incombination with a Cancer-On-Chip recapitulate the in vivo process whereimmune cells are recruited from and/or educated in the above organsbefore reaching the tumor site. In one embodiment, linking Lymph node-to the Cancer-On-Chip is to recapitulate the full process includingantigen presentation, T-cell education and recruitment. Similarly,linking of Bone marrow to the Cancer-On-Chip is done to recreate theenvironment promoting proliferation, differentiation and recruitment tothe tumor of myeloid-derived cells.

In one embodiment, a Thymus-on-Chip is linked to a Cancer-On-Chip. Inone embodiment, in vivo relevant DC-tumor cell interactions arecontemplated, in part for proceeding with the incorporation oflymphocytes and experimentation with immunotherapies. In one embodiment,tumor dendritic cells (DC) are added in order to assess their priming byexposure to tumor antigens.

Interaction of the Immune System with the Cancer-On-Chip, e.g. formodeling immunocyte migration in the tumor; Perfuse dendritic cells (DC)into the tumor in order to prime them with tumor-specific antigens andexpose a separately maintained culture of fluorescently labeledcytotoxic cells to the primed DC to assess their interaction. Thesecytotoxic cells will then be perfused into the chip through the“vascular” channel to assess the existence of an in vivo relevant“immune privileged” environment near the tumor. These developments willenable development of a model for the recruitment of immune cells by thetumor.

E. Interaction of Dendritic Cells with the Cancer-On-Chip

The goal is to obtain the in vivo relevant DC-tumor cell interaction,required for proceeding with the incorporation of lymphocytes andexperimentation with immunotherapies. As our strategy includes gradualdevelopment of the tumor immune environment first we will flow throughthe tumor dendritic cells (DC) in order to assess their priming byexposure to tumor antigens. This is a critical step, as it will help toconfirm recapitulation of functional interactions critical in vivo. ThePOC in mouse Tumor-Chip in this step may help in the engineering andfine tuning of the system, to proceed with the human cells and providesthe in vivo correlation.

IV. Examples of Cancer-On-Chip Embodiments for, e.g., Identification ofTargets for Testing Cancer Therapeutics, Cancer Prevention, orMetastasis Prevention

A. Tumor-Introduction Cancer-On-Chip

In some embodiments, a Cancer-On-Chip is a Tumor-IntroductionCancer-On-Chip. A Tumor-Introduction Cancer-On-Chip refers to chipsinitially having no tumor cells, and either cells or microenvironmentelements from a) a non-cancer microenvironment (e.g. normal healthycells, or cells away from the site of a tumor), b) the site of a tumor(or in close proximity), or c) an environment with a pre-disposition orrisk factor for cancer (e.g. derived from a patient with knownsusceptibility alleles). Such cells or microenvironment elements mayinclude epithelial cells, stromal cells, immune cells, connectivetissues, ECM, soluble factors, lamina propria derived cells (LPDC), etc.In one aspect of the invention, a tumor-generating process is enacted onthe Tumor-Introduction Cancer-on-Chip. For example, tumor cells may beintroduced into the Cancer-on-Chip's parenchymal compartment (e.g. byflowing in tumor cells), tumor cells may be introduced into theCancer-on-Chip's vascular compartment (this may serve as a model ofmetastasis), by the application of radiation, and/or by the introductionof a carcinogenic agent. The Tumor-Introduction Cancer-on-Chip can beused, for example, to help determine whether an agent (e.g. apharmaceutical compound) may protect from tumor introduction or whetherany of the Cancer-on-Chip's elements are protective or act as riskfactors, and if so, by what mechanisms. In such embodiments, theCancer-on-Chip may provide utility in identifying the significance ofdifferent elements of the cancer microenvironment or patient background.For example, in one embodiment, we disclose a method wherein two or moreCancer-on-Chips are compared, wherein two or more of the Cancer-on-Chipsdiffer in the origin of one or more of its components (e.g. particularcells, ECM, soluble factors or other components originate from the siteof a tumor vs. away from it, or from an at-risk patient vs. anormal-risk patient, etc.)

B. Sick and at-Risk Cancer-On-Chip

In contrast to the Tumor-Introduction Cancer-On-Chip, in someembodiments, a Cancer-On-Chip is a Sick Cancer-On-Chip. A SickCancer-On-Chip refers to a chip containing pre-cancerous cells orcancerous cells. In some embodiments of the Sick Cancer-on-Chip, whichwe Willi the At-Risk Sick Cancer-on-Chip may comprise cells and/orcomponents isolated from a cancerous microenvironment, such asepithelial cells, endothelial cells, stromal cells, stroma, immunecells, tissues, ECM (e.g. complete or an isolated component), solublefactors, lamina propria derived cells (LPDC), etc., derived fromsubjects with risk factors for the type of cancer for use in the chip,cells derived from subjects known to have risk factors for any type ofcancer, cells derived from subjects known to have or under treatment forcancer or under treatment for another disease where a side effect is thedevelopment of cancer cells or be in remission from cancer, e.g. cellsor cells derived from subjects known susceptibility alleles for the typeof cancer for use in the chip, etc. In other words, a At-Risk SickCancer-On-Chip refers to chips where at least one of the components areknown to have genetic or physiological association with cancer arisingfrom at least one sick component on a Sick Cancer-On-Chip. In someembodiments, such a Sick Cancer-On-Chip with at least one sick componentis used to determine stages of cancer development in response toendogenous factors. In some embodiments, such a Sick Cancer-On-Chip withat least one sick component is used to determine stages of cancerdevelopment in response to simulated exogenous agents, including, forexample, drugs, chemotherapy, suspected or known carcinogens,immunotherapy, or antibodies added to or flowed through the SickCancer-On-Chip.

C. Metastatic System Comprising Multiple Cancer-On-Chips

Certain types of cancers are associated with metastasis to other organswhere secondary cancer/tumors grow. As on example, common sites ofmetastases for lung cancer are other parts of the lung, adrenal gland,bones, brain and liver. Thus, in one embodiment, a Cancer-on-Chipcomprises tumor cells from one organ-type disposed within or introducedto an Organ-Chip of a different organ-type.

In another embodiment, a Cancer-On-Chip effluent (outflow) isfluidically linked (i.e. in fluidic communication) with anotherCancer-On-Chip. As one example, a first Cancer-On-Chip is fluidicallylinked with a second Organ-Chip, with the second Organ-Chip representingthe same organ-type as the first Cancer-on-Chip. Such embodiment can beused, for example, for identifying factors and/or testing factors ortesting anti-metastatic agents on cancer cell metastasis from one tissuesite (e.g. microenvironment) to another tissue site (e.g.microenvironment) within the same tissue or organ.

As another example, a first Cancer-On-Chip effluent (outflow) isfluidically linked (in fluidic communication) with a second Organ-Chip,with the second Organ-Chip representing a different organ-type than thefirst Cancer-on-Chip. Such embodiment can be used, for example, foridentifying factors and/or testing factors or testing antimetastaticagents on metastasis from one type of tissue or organ to another tissueor organ. Thus, in some embodiments, the first Cancer-On-Chip is a LungCancer-On-Chip while the second chip may be any one or more of, merelyfor non-limiting examples, an Adrenal gland-On-Chip, BoneMarrow-On-Chip, Brain-On-Chip and liver-On-Chip.

As one example, colon cancer is associated with metastatic secondaryliver cancer. Thus, in one embodiment, a colon Cancer-On-Chip effluent(outflow) is fluidically linked to a liver-On-Chip. As another example,colon cancer is associated with metastatic secondary lung cancer.

Thus, in one embodiment, a colon Cancer-On-Chip effluent (outflow) isfluidically linked to a Lung-On-Chip. As yet another example, pancreaticcancer is associated with metastatic secondary liver cancer. Thus, inone embodiment, a pancreatic Cancer-On-Chip effluent (outflow) isfluidically linked to a Liver-On-Chip.

In some embodiments, various organ chip devices described in theInternational Patent Application Nos. PCT/US2009/050830;PCT/US2012/026934; PCT/US2012/068725; PCT/US2012/068766;PCT/US2014/071611; and PCT/US2014/071570, the contents of each of whichare incorporated herein by reference in their entireties, can bemodified to form the devices described herein. For example, the organchip devices described in those patent applications can be modified inaccordance with the devices described herein.

In yet further embodiments, a Sick Cancer-On-Chip is fluidically linkedto (in fluidic communication with) a Tumor-Introduction Cancer-On-Chip,wherein cancer cells for seeding a Tumor-Introduction Cancer-On-Chipderive from metastatic cells, e.g. cells actively detaching from a SickCancer-On-Chip flowing to a Tumor-Introduction Cancer-On-Chip or shedfrom a tumor growing in a Sick Cancer-On-Chip. As one example, fluidflowing through microfluidic connections to a Sick Cancer-On-Chip flowsinto to a Tumor-Introduction Cancer-On-Chip. In some embodiments,microfluidic connections may be coated with a material so that flowingcells do not stick to or become attached to internal surfaces of thefluidic connections. In some embodiments, microfluidic connections maybe coated with a material so that metastatic cells may migrate bycrawling along internal surfaces of the fluidic connections into thenext chip.

In some embodiments, one or more of the fluidic connection between theaforementioned microfluidic devices (e.g. first Cancer-on-Chip andsecond Organ-Chip) may comprise tube, channels or bridges. In otherembodiments, the said one or more fluidic connections may comprisediscrete fluid transfers. Such discrete transfers may be enactedmanually or by means or a liquid-handling robot or autosampler.

V. Testing Immunotherapeutics in the Tumor and Immune System-Chips

In one contemplated embodiment, demonstration that Chips canrecapitulate response and effects on the tumor tissue and immune systemto known therapeutics, such as check-point inhibitors currently inclinical use. Thus, the Cancer-on-Chips technology can serve as aplatform for testing of novel therapeutics with the ability to predictefficacy, toxicities, and mechanism of action in an in vivo relevant,dynamic human cells environment. Further, use the Cancer-on-Chipssystems to support a system biology approach to discover new potentialtargets and biomarkers for therapeutic development.

As one example, Human B7 homolog 1 (B7-H1), also called programmed celldeath 1 ligand 1 (PDCD1L1) and programmed death ligand 1 (PD-L1), is amember of the growing B7 family of immune proteins that are understoodto provide signals for both stimulating and inhibiting T cellactivation. Without being bound by theory, PD-L1 binds to PD-1, which isexpressed on the surface of activated T cells. The formation of a PD-1receptor/PD-L1 ligand complex transmits an inhibitory signal whichreduces the proliferation of T cells. These ligands are regarded asendogenous “checkpoints” for the immune system that allow fortermination of an immune response after antigen activation (e.g. from aninfection).

There is strong evidence that many tumors utilize these immunecheckpoint molecules to evade immune destruction. Tumors have been shownto express PD-L1 as a soluble factor and/or on their surface. Theseobservations have resulted in intensive efforts to developimmunotherapeutic approaches for cancer, includingimmune-checkpoint-pathway inhibitors such as human antibodies. In oneembodiment, these checkpoint inhibitors are introduced into themicrofluidic device comprising cancer cells (Cancer-on-chip) and theresults are detected and measured.

The formation of a PD-1 receptor/PD-L1 ligand complex can be blocked byan antibody to either ligand. Thus, for cancer, one might block the PD-1receptor on T cells, or one might block the PD-L1 ligand in solution oron the tumor cell. While one might think targeting either ligandgenerates comparable results, the clinical trial data (discussed below)shows some differences.

These ligands are not the only checkpoint molecules. CTLA-4 is anothersuch immune molecule. As discussed below, BMS has commercialized ananti-CTLA-4 antibody (ipilimumab) for the treatment of patients withadvanced melanoma.

In some contemplated embodiments, use Cancer-on-Chips technology toanswer specific questions including determining the relevance ofpathways/targets elucidated in mice, in humans, mechanism of action, andelucidating of novel mechanisms for drug targeting and immunotherapeuticdevelopment. Some examples of questions we could address would include:Address the potential impact of specific immunotherapy strategies onimmune cells chemotaxis and tumor invasion and the corresponding changesin tumor biology; Study the effect of immunotherapy on tumor angiogenicactivity; Study the effects of novel check-point inhibitors and relevantbiologics and/or small molecules targeting the immune/tumor cellsinteraction;

Employ patient-specific tumor cells and reconstitute the wholecancer-on-chip with patient-specific cells, which may be primary cellsand/or iPS-derived cells, including but not limited to endothelial,fibroblasts and/or immune cells. iPS cells that are differentiated tocell such as neurons, endothelial cells, hepatocytes, lung and gutepithelial cells may be used in Cancer-on-Chips; contribution ofmicrobiome in tumor expansion or regression, as a prototype for studyingmicrobiome-primary human tissue symbiosis and functional interaction;and efficacy of immune cells to attack the metastatic tumor, such as forexample via interconnected Gut-on-Chips and Hepatoma Chips

In some embodiments, the present invention contemplates usingCancer-on-Chips technology to trace the metastatic potential of tumors.Through effluent transfer we could explore the potential for circulatingtumor cells to develop new cancer lesions in distant organs. Potentialstudies could then include: Manipulation of the Chip microenvironment totest impact of these changes on metastatic potential of the tumor. Thiscould also be applied to patient-specific chips for precision medicineapplications. Current microfluidic-based approaches for studyingcirculating tumor cells are too simplistic and therefore do not have therequired biological complexity that Organ Cancer-on-Chips provide.Cancer-on-Chips could provide a useful tool to understand the mechanismsfor lack of efficacy of many oncology drugs, uncovered often only inphase 2 clinical trials, as well as, address recent safety concerns suchas with CAR T cell therapy, or other modified, engineered or activatedimmune cells used for immunotherapies.

Although in recent clinical trials, CAR T cell therapy has dramaticallyimproved the outcomes of blood cancer patients with advanced, otherwiseuntreatable, forms of leukemia and lymphoma, the full potential of CARsfor treating solid tumors has not been reached and many challengesremain. Having more predictive, human relevant systems to study humantumor biology, species difference in tumor biology between mice andhumans, as well as the interactions of the human immune system with thetumor would advance our knowledge and help to provide the most robustand precise preclinical platforms for drug discovery for thisdevastating disease and enable the advancement of immunotherapies.

VI. Endpoints and Analysis Using the Chips

In some contemplated embodiments, Cancer-on-Chips may be evaluated byassays, including but not limited to: RNA and micro RNA profiling;Biochemical assays; Clinical chemistry panels; Metabolomic analysis;Proetomic analysis; Epigenomic profiling; Biomarkers; Imaging andhistology; ELISAs; electrochemical sensing; mass spectrometry; and Flowcytometry.

VII. Potential Applications of Cancer-On-Chips

In some contemplated embodiments, Cancer-on-Chips may be used forMaintenance of long-term viability and function (weeks/months);High-resolution, real-time imaging; In vitro analysis of biochemical,genetic, and metabolic activities; Ability to step-wise recreate tissuecomplexity in vitro (introduction of multiple cell types into the systemin relevant architecture); Engineering provides fine control over themicroenvironment including mechanical forces and ECM; Able to studyreal-time complex cellular interactions not possible in animal models;Fluidic nature of the system allows linking and interactions betweendifferent organs systems (e.g. Lymph node-on-Chip and Tumor-on-Chip) notpossible with other in vitro systems; Flow in the system allows analysisof recruitment of circulating immune cells that is central to theetiology of many diseases and toxicities; Flow creates a dynamic systemwith fresh nutrients that recapitulate circulation. The dynamic natureof systems provides an opportunity for improvedpharmacokinetic/pharmacodynamic predictions; Enable mechanism of actionstudies in physiologically relevant system, e.g. for furtherunderstanding of mechanistic interaction between immune and tumor cellsin a human relevant system, that will more accurately translate to theclinic; Can complement existing animal models and provide mechanisticinsight and a bridge between existing animal models and translation tohumans; Enable a systems biology approach for example to identify newpotential targets for therapeutic development or biomarkeridentification. E.g. may uncover the order that specific interactionsand pathways need to be targeted for the elimination of the tumor, aswell as highlight the most critical nodes in this process. Enablediscovery of novel targets for cancer immunotherapies; Enableidentification off target effect and potential safety liabilities;Identification of novel biomarkers for assessment of clinical efficacyof cancer immunotherapies; Facilitate progress in personalized/precisionmedicine, with the potential to in the future use the Organs-on-Chips ina diagnostic application. This strategy may provide a unique approach toassess personalized immunotherapy and precision medicine in oncology,major goal of the field, as an effective anti-tumor therapeuticstrategy; The power of the data obtained from these models can also beincrease by combining with other efforts such humanized animal models,clinical trial data GWAS studies, single cells analysis; TheCancer-on-Chips have the potential to contribute to the extensiveefforts to improve understanding of cancer biology and immune systeminteractions by providing a platform to test tumor and patient-specifictherapeutic modalities and improve translation of animal data to theclinic.

Advantages of using Cancer-on-Chips, in additional to the high level ofbiological function and complexity achieved, is that an entire systemthat will include the Cancer-on-Chips coupled with the appropriateinstrumentation and software, a Human Cancer Emulation System.

This will enable the end users in the future to easily employ thetechnology in their labs without any prior engineering expertise, priorexperience handling Cancer-on-Chips, or specific know how.

DETAILED DESCRIPTION OF THE INVENTION

A Cancer-On-Chip (16) is composed of a clear, flexible polymer about thesize of a USB memory stick, containing hollow fluidic channels (1) linedby living cells (see FIG. 4). These Cancer-On-Chips fully recreate thecomplex, dynamic state in which a living cancer cell interacts with andfunctions within a real human organ: including but not limited to havingsubstrate (extracellular matrix), tissue-tissue interface and relevantepithelial, endothelial interactions, mechanical forces, immune cellsand blood components, and biochemical surroundings. The fluidic natureof the system allows not only the recreation of mechanical forcesapplied by normal flow of blood in the body (sheer stress) known to becritical for both endothelial and epithelial cell function as well asother mechanical forces such as that cells experience in the body fromsay stretching of alveolar lung tissue as we breath. There issignificant literature that demonstrates the importance of mechanicalforces and mechanostransduction in biology and disease development—fromdetermining cell shape and cell-cell interaction, changing geneexpression profiles, to playing pivotal role in development biology anddisease pathophysiology. The ability to recapitulate in vivo relevantmechanical forces in vitro is a feature that is missing from other invitro systems and a clear advantage of our approach—specifically instudying tumor microenvironment. The fluidic nature of the systemfurther allows the emulation of the dynamic environment that exists intissues and organs and it further provides the ability to link differentCancer-On-Chips together to emulate the organ-to-organ interactionsoccurring in vivo providing a window into the physiology and improvedmechanistic insight into human diseases and drug responses.

The microchannel(s) in the microfluidic devices can be substantiallylinear or they can be non-linear. In some embodiments, the channels arenot limited to straight or linear channels and can comprise curved,angled, or otherwise non-linear channels. It is to be further understoodthat a first portion of a channel can be straight, and a second portionof the same channel can be curved, angled, or otherwise non-linear.Without wishing to be bound by a theory, a non-linear channel canincrease the ratio of culture area to device area, thereby providing alarger surface area for cells to grow. This can also allow for a higheramount or density of cells in the channel. In some embodiments, thedevice can comprise an inlet channel connecting an inlet fluid port tothe first chamber. The inlet channels and inlet ports can be used tointroduce cells, agents (e.g., but not limited to, stimulants, drugcandidate, particulates), airflow, and/or cell culture media into thefirst chamber.

I. A Membrane Located in Between the First Structure and SecondStructure.

In one embodiment, the membrane is oriented along a plane between thefirst chamber and the second chamber. It should be noted that althoughone membrane is typically used, more than one membrane can be configuredin devices which comprise more than two chambers.

The membrane separating the first chamber and the second chamber in thedevices described herein can be porous (e.g., permeable or selectivelypermeable), non-porous (e.g., non-permeable), rigid, flexible, elasticor any combinations thereof. Accordingly, the membrane can have aporosity of about 0% to about 99%. As used herein, the term “porosity”is a measure of total void space (e.g., through-holes, openings,interstitial spaces, and/or hollow conduits) in a material, and is afraction of volume of total voids over the total volume, as a percentagebetween 0 and 100% (or between 0 and 1). A membrane with substantiallyzero porosity is non-porous or non-permeable.

As used interchangeably herein, the terms “non-porous” and“non-permeable” refer to a material that does not allow any molecule orsubstance to pass through.

In some embodiments, the membrane can be porous and thus allowmolecules, cells, particulates, chemicals and/or media to migrate ortransfer between the first chamber and the second chamber via themembrane from the first chamber to the second chamber or vice versa.

As used herein, the term “porous” generally refers to a material that ispermeable or selectively permeable. The term “permeable” as used hereinmeans a material that permits passage of a fluid (e.g., liquid or gas),a molecule, a whole living cell and/or at least a portion of a wholeliving cell, e.g., for formation of cell-cell contacts. The term“selectively permeable” as used herein refers to a material that permitspassage of one or more target group or species, but act as a barrier tonon-target groups or species. For example, a selectively-permeablemembrane can allow passage of a fluid (e.g., liquid and/or gas),nutrients, wastes, cytokines, and/or chemokines from one side of themembrane to another side of the membrane, but does not allow wholeliving cells to pass through. In some embodiments, aselectively-permeable membrane can allow certain cell types to passthrough but not other cell types.

In some embodiments, a membrane can be a hydrogel or a gel comprising anextracellular matrix polymer, and/or a biopolymer or biocompatiblematerial. In some embodiments, the hydrogel or gel can be embedded witha conduit network, e.g., to promote fluid and/or molecule transport.See, e.g., Wu et al. (2011) “Omnidirectional Printing of 3DMicrovascular Networks.” Advanced Materials 23: H178-H183; and Wu et al.(2010) “Direct-write assembly of biomimetic microvascular networks forefficient fluid transport.” Soft Matter 6: 739-742, for example methodsof introducing a conduit network into a gel material.

In some embodiments, a porous membrane can be a solid biocompatiblematerial or polymer that is inherently permeable to at least onematter/species (e.g., gas molecules) and/or permits formation ofcell-cell contacts. In some embodiments, through-holes or apertures canbe introduced into the solid biocompatible material or polymer, e.g., toenhance fluid/molecule transport and/or cell migration. In oneembodiment, through-holes or apertures can be cut or etched through thesolid biocompatible material such that the through-holes or aperturesextend vertically and/or laterally between the two surfaces of themembrane. It should also be noted that the pores can additionally oralternatively incorporate slits or other shaped apertures along at leasta portion of the membrane which allow cells, particulates, chemicalsand/or fluids to pass through the membrane from one section of thecentral channel to the other.

In some embodiments, pillars can be used instead of (or together with) amembrane. The spacing and dimensions of the pillars can be adjusted topermit or block the passage of cells.

In some embodiments, the membrane can be coated with substances such asvarious cell adhesion promoting substances or ECM proteins, such asfibronectin, laminin, various collagen types, glycoproteins,vitronectin, elastins, fibrin, proteoglycans, heparin sulfate,chondroitin sulfate, keratin sulfate, hyaluronic acid, fibroin,chitosan, or any combinations thereof. In some embodiments, one or morecell adhesion molecules can be coated on one surface of the membrane 208whereas another cell adhesion molecule can be applied to the opposingsurface of the membrane 208, or both surfaces can be coated with thesame cell adhesion molecules. In some embodiments, the ECMs, which canbe ECMs produced by cells, such as primary cells or embryonic stemcells, and other compositions of matter are produced in a serum-freeenvironment.

In an embodiment, one can coat the membrane with a cell adhesion factorand/or a positively-charged molecule that are bound to the membrane toimprove cell attachment and stabilize cell growth. The positivelycharged molecule can be selected from the group consisting ofpolylysine, chitosan, poly(ethyleneimine) or acrylics polymerized fromacrylamide or methacrylamide and incorporating positively-charged groupsin the form of primary, secondary or tertiary amines, or quaternarysalts. The cell adhesion factor can be added to the membrane and isfibronectin, laminin, various collagen types, glycoproteins,vitronectin, elastins, fibrin, proteoglycans, heparin sulfate,chondroitin sulfate, keratin sulfate, hyaluronic acid, tenascin,antibodies, aptamers, or fragments or analogs having a cell bindingdomain thereof. The positively-charged molecule and/or the cell adhesionfactor can be covalently bound to the membrane. In another embodiment,the positively-charged molecule and/or the cell adhesion factor arecovalently bound to one another and either the positively-chargedmolecule or the cell adhesion factor is covalently bound to themembrane. Also, the positively-charged molecule or the cell adhesionfactor or both can be provided in the form of a stable coatingnon-covalently bound to the membrane.

In some embodiments, cells are cultured on and/or under the membraneunder flow conditions. In some embodiments, there is a steady-stateperfusion of the cells. In other embodiments described herein, thedevices can comprise a flowing culture medium in the first chamberand/or the second chamber, wherein the flowing culture medium generatesa shear stress. Based on the viscosity of the culture medium and/ordimensions of the chambers, one of skill in the art can determineappropriate flow rates of culture medium through the chambers to achievedesired shear stress. In some embodiments, the flow rate of the culturemedium through the first chamber can range from about 5 μL/hr to about50 μL/hr. In some embodiments, the flow rate of the culture mediumthrough the second chamber can range from about 15 μL/hr to about 150μL/hr.

Thus, in one embodiment, fluidic shear forces are generated. In someembodiments, the first chamber, the second chamber or both may compriseor be in communication with one or more fluidic channels. Such channelsmay allow, for example, the perfusion, the delivery or removal ofreagents, and/or the collection of samples from one or both of thechambers. Such channels may provide independent fluidic access to eachchamber, and correspondingly, to either side of the membrane.

II. Optional Mechanical Actuation and Vacuum Channels

In some embodiments, the microfluidic devices comprises a means forcreating mechanical actuation. Such mechanical actuation has beendemonstrated to enact a biological effect, which may improve theemulation of the in vivo environment (REF: original Lung-on-a-Chippaper). Several designs for the mechanical actuation of Organ-Chips aredisclosed in REF (HU4868). In some embodiments, fluidic channels indevices of the present inventions are optionally flanked by two vacuumchannels that allow the pneumatically actuated stretching forcesmimicking intestinal peristalsis. In some embodiments, stretching forcesare for stretching an epithelial layer. In one embodiment, mechanicalforces are generated.

III. Optional Gels

In some embodiments, the microfluidic device comprises a gel. Such a gelmay provide an additional culture compartment, which may be used, forexample, for culturing cells embed within the gel. Moreover, the gel maybe invaded, reshaped, or remodeled by cells, thereby allowing themicrofluidic to emulate various phenomena that occur in vivo.

III. Open Top Microfluidic Cancer-On-Chips.

The present disclosure relates to Cancer-On-Chips, such as fluidicdevices comprising one or more cells types for the simulation one ormore of the function of bodily components, e.g. normal cells, cancercells, cells derived from a tissue area, cells derived from blood, cellsderived from an organ at risk of developing cancer, or a component of amicroenvironment derived from an area where cancer cells arose butwithout cancer cells, etc. Accordingly, the present disclosureadditionally describes open-top Cancer-On-Chips, see, e.g. schematic inFIGS. 4, 11-12. FIG. 11 shows an exemplary exploded view of oneembodiment of an open-top chip device 1800, wherein a membrane 1840resides between the bottom surface of the first chamber 1863 and thesecond chamber 1864 and the at least two spiral microchannels 1851. Opentop microfluidic chips include but are not limited to chips havingremovable covers, such as removable plastic covers, paraffin covers,tape covers, etc.

Many of the problems associated with earlier systems can be solved byproviding an open-top style microfluidic device that allows topicalaccess to one or more parts of the device or cells that it comprises.For example, the microfluidic device can include a removable cover, thatwhen removed, provides access to the cells of interest in themicrofluidic device. In some aspects, the microfluidic devices includesystems that constrain fluids, cells, or biological components todesired area(s). The improved systems provide for more versatileexperimentation when using microfluidic devices, including improvedapplication of treatments being tested, improved seeding of additionalcells, and/or improved aerosol delivery for select tissue types.

It is also desirable in some aspects to provide access to regions of acell-culture device. For example, it can be desirable to provide topicalaccess to cells to (i) apply topical treatments with liquid, gaseous,solid, semi-solid, or aerosolized reagents, (ii) obtain samples andbiopsies, or (iii) add additional cells or biological/chemicalcomponents

Therefore, the present disclosure relates to fluidic systems thatinclude a fluidic device, such as a microfluidic device with an openingthat provides direct access to device regions or components (e.g. accessto the gel region, access to one or more cellular components, etc.).Although the present disclosure provides an embodiment wherein theopening is at the top of the device (referred to herein with the term“open top”), the present invention contemplates other embodiments wherethe opening is in another position on the device. For example, in oneembodiment, the opening is on the bottom of the device. In anotherembodiment, the opening is on one or more of the sides of the device. Inanother embodiment, there is a combination of openings (e.g. top andsides, top and bottom, bottom and side, etc.).

While detailed discussion of the “open top” embodiment is providedherein, those of ordinary skill in the art will appreciate that manyaspects of the “open top” embodiment apply similarly to open bottomembodiments, as well as open side embodiments or embodiments withopenings in any other regions or directions, or combinations thereof.Similarly, the device need not remain “open” throughout its use; rather,as several embodiments described herein illustrate, the device mayfurther comprise a cover or seal, which may be affixed reversibly orirreversibly. For example, removal of a removable cover creates anopening, while placement of the cover back on the device closes thedevice. The opening, and in particular the opening at the top, providesa number of advantages, for example, allowing (i) the creation of one ormore gel layers for simulating the application of topical treatments onthe cells, tissues, or organs, or (ii) the addition of chemical orbiological components such as the seeding of additional cell types forsimulated tissue and organ systems. The present disclosure furtherrelates to improvement in fluidic system(s) that improve the delivery ofaerosols to simulated tissue and organ systems, such as simulatedgastrointestinal tissues.

The present invention contemplates a variety of uses for these open topmicrofluidic devices and methods described herein. In one embodiment,the present invention contemplates a method of topically testing anagent (whether a drug, gas, or other substance) comprising 1) providinga) an agent and b) microfluidic device comprising i) a chamber, saidchamber comprising a lumen and projections into the lumen, said lumencomprising ii) a gel matrix anchored by said projections and comprisingcells (e.g. tumor cells) in, on or under said gel matrix, said gelmatrix positioned above iii) a porous membrane and under iv) a removablecover, said membrane in contact with v) fluidic channels; 2) removingsaid removable cover; and 3) topically contacting said cells in, on orunder said gel matrix with said agent. In one embodiment, said agent isin an aerosol. In one embodiment, agent is in a liquid, gas, gel,semi-solid, solid, or particulate form. These uses may apply to the opentop microfluidic chips described below and herein.

A. Open Top Microfluidic Chips without Gels.

In one embodiment, open top Cancer-On-Chips do not contain gels, eitheras a bulk gel or a gel layer. Thus, the present invention alsocontemplates, in one embodiment, a layered structure comprising i)fluidic channels covered by ii) a porous membrane, said membranecomprising iii) a layer or collection of cells (e.g. tumor cells) andsaid membrane positioned below said cells. In one embodiment, there is aremovable cover over the cells.

Additional embodiments are described herein that may be incorporatedinto open top chips without gels.

B. Open Top Microfluidic Chips with Gels.

Furthermore, the present disclosure contemplates improvements to fluidicsystems that include a fluidic device, such as a microfluidic devicewith an open-top region that reduces the impact of stress that can causethe delamination of tissue or related component(s) (e.g., such as a gellayer). Thus, in a preferred embodiment, the open-top microfluidicdevice comprises a gel matrix. In one embodiment, the open-topmicrofluidic device does not contain a bulk gel.

The present invention also contemplates, in one embodiment, a layeredstructure comprising i) fluidic channels covered by ii) a porousmembrane, said membrane comprising iii) a layer or collection of cells(e.g. tumor cells) and said membrane positioned below iv) a gel matrix.In one embodiment, there is a removable cover over the gel matrix(and/or cells). It is not intended that the present invention be limitedto embodiments with only one gel or gel layer. In one embodiment, thelayered structure further comprises a second gel matrix (e.g. positionedunder said membrane). The gel(s) or coatings can be patterned or notpatterned. Moreover, when patterned, the pattern need not extend to theentire surface. For example, in one embodiment, at least a portion ofsaid gel matrix is patterned. It is not intended that the presentinvention be limited by the nature or components of the gel matrix orgel coating. In one embodiment, gel matrix comprises collagen. A varietyof thickness is contemplated. In one embodiment of the layeredstructure, said gel matrix is between 0.2 and 6 mm in thickness.

In yet another embodiment, the present invention contemplates amicrofluidic device comprising i) a chamber, said chamber comprising alumen and projections in the lumen, said lumen comprising ii) a gelmatrix anchored by said projections, said gel matrix positioned aboveiii) a porous membrane, said membrane in contact with iv) fluidicchannels. In one embodiment, said membrane comprises cells (e.g. tumorcells). The projections serve as anchors for the gel. The projections,in one embodiment, project outward from the sidewalls. The projections,in another embodiment, project upward. The projects, in anotherembodiment, project downward. The projections can take a number of forms(e.g. a T structure, a Y structure, a structure with straight or curvingedges, etc.). In some embodiments, there are two or more projections; inother embodiments, there are four or more projections to anchor the gelmatrix. In one embodiment, the membrane is above said fluidic channels.

In other embodiments, open top microfluidic chips comprise partiallumens as described herein for closed top chips. Thus, in someembodiments, open top microfluidic chips comprise lumens formed byviscous fingering described herein for closed top chips.

Lumen gel structures may be used in several types of embodiments foropen top microfluidic chips, e.g. epithelial cells can be attached tooutside of the gel, or within the gel. In some embodiments, ECMcomponents may be added within the gel, or below the gel. In someembodiments, LPDCs may be added within the gel, or below the gel. Insome embodiments, stomal cells are added within the gel. In someembodiments, stomal cells are attached to the side of the gel oppositefrom the lumen. In some embodiments, endothelial cells are located belowthe gel on the side opposite the lumen. In some embodiments, endothelialcells may be present within the gel. Additional embodiments aredescribed herein that may be incorporated into open top chips with gels.

The present invention contemplates combining features from differentembodiments. The present invention contemplates removing features fromthe above-indicated embodiments. For a non-limiting example, co-culturesof cancer cells with epithelial cells, endothelial cells and stromalcells may have a feature removed. For example, subsets of cells isolatedfrom infiltrates of cancer cells, such as TILs, may be removed from theconfiguration in order to identify cells or components, contributing tospecific disease phenotypes. For another non-limiting example, stromalcells may be removed from the configuration in order to identifycomponents contributing to specific disease phenotypes. The presentinvention contemplates adding features to the configuration in order toidentify cells initiating a specific cancer phenotype, e.g. addingdiseased LP-derived cells, e.g. isolated from areas of tumor growth, tomicrofluidic devices containing precancerous or healthy cells. Thepresent invention contemplates substituting features in theabove-indicated embodiments. For a non-limiting example, ECM fromcommercial sources may be substituted with ECM isolated from humans.

IV. Exemplary Devices for Simulating a Function of a Tissue.

Some embodiments described herein relate to devices for simulating afunction of a tissue, in particular a gastrointestinal tissue. In oneembodiment, the device generally comprises (i) a first structuredefining a first chamber; (ii) a second structure defining a secondchamber; and (iii) a membrane located at an interface region between thefirst chamber and the second chamber to separate the first chamber fromthe second chamber, the membrane including a first side facing towardthe first chamber and a second side facing toward the second chamber.The first side of the membrane may have an extracellular matrixcomposition disposed thereon, wherein the extracellular matrix (ECM)composition comprises an ECM coating layer. In some embodiments, an ECMgel layer e.g. ECM overlay, is located over the ECM coating layer.

V. ECM Coating.

To determine optimum conditions for cell attachment, the surface-treatedmaterial (e.g., APTES-treated or plasma-treated PDMS) can be coated withan ECM coating of different extracellular matrix molecules at varyingconcentrations (based on the resulting cell morphology and attachment).

VI. ECM Overlay.

The ECM overlay is typically a “molecular coating,” meaning that it isdone at a concentration that does not create a bulk gel. In someembodiments, an ECM overlay is used. In some embodiments, an ECM overlayis left in place throughout the co-culturing. In some embodiments, anECM overlay is removed, e.g. when before seeding additional cells into amicrofluidic device. In some embodiments, the ECM layer is provided bythe cells seeded into the microfluidic device.

Although cells described for use in a Cancer-On-Chip make their own ECM,it is contemplated that ECM in predisease and diseased states may may befound in areas around sites of cancer cell growth. Further, the proteinmicroenvironment provided by ECM also affects cells. Thus it iscontemplated that tissue-derived ECM may carry over a disease state.Therefore, in addition to the ECM described herein, ECM used inmicrofluidic devises of the present inventions may be derived from orassociated with areas in and around sites of cancer cells. In oneembodiment, a device comprising tissue-derived ECM may be used asdescribed herein, to identity contributions to healthy or disease statesaffected by native ECM.

For example, ECM may be isolated from biopsies of healthy, non-diseaseand disease areas as tissue-derived ECM. Isolates for use may includecells within or attached or further processed to remove embedded cellsfor use in the absence of the cells.

Additional examples of ECM materials include but are not limited toMatrigel®, Cultrex®, ECM harvested from humans, etc.

Matrigel® is a trade name for a solubilized basement membranepreparation extracted from the Engelbreth-Holm-Swann (EHS) mousesarcoma, a tumor rich in such ECM proteins as laminin (a majorcomponent), collagen IV, heparin sulfate proteoglycans,entactin/nidogen, and a number of growth factors as produced andmarketed by Corning Life Sciences. Matrigel® gels to form areconstituted basement membrane. Versions of Matrigel® include BDMatrigel® (Basement Membrane) Matrix, offered as Standard, Growth FactorReduced, Growth Factor Reduced-High Concentration (HC) and Growth FactorReduced-Phenol Red-Free formulations, BD Matrigel® hESC-qualifiedMatrix, by BD Biosciences.

Trevigen, Inc. markets other ECM versions of BME harvested as a solubleform of basement membrane purified from Engelbreth-Holm-Swarm (EHS)tumor cells under the trade name Cultrex® Basement Membrane Extract(BME). Cultrex® extract gels at 37° C. to form a reconstituted basementmembrane. The major components of Cultrex® BME include laminin, collagenIV, entactin, and heparin sulfate proteoglycan. Several forms Cultrex®are offered by Trevigen as: Cultrex® Reduced Growth Factor BasementMembrane Extract, Type R1. Type R1 matrix provides a proprietaryformulation that has higher tensile strength when compared to otherCultrex® products, i.e. Cultrex® BME, Cultrex® BME Type 2 and Cultrex®BME Type 3. Type R1 has a higher concentration of entactin, one of theBME components that connects laminins and collagens reinforcing thehydrogel structure. Cultrex® BME Type R1 has been specifically designedto culture tissue organoids. BME type R1 supports culture of humangastric or small intestine organoids. In a Tube formation assay—BME typeR1 promotes formation of capillary-like structures by human (HBMVEC;HUVEC); Barker, et al., Lgr5+ve Stem Cells Drive Self-Renewal in theStomach and Build Long-Lived Gastric Units In Vitro. Cell Stem Cell,2010. 6(1): p. 25-36; Sato, T., et al., Single Lgr5 stem cells buildcrypt-villus structures in vitro without a mesenchymal niche. Nature,2009. 459(7244): p. 262-26; Sato, T. and H. Clevers, GrowingSelf-Organizing Mini-Guts from a Single Intestinal Stem Cell: Mechanismand Applications. Science, 2013. 340(6137): p. 1190-1194; Jung, P., etal., Isolation and in vitro expansion of human colonic stem cells. NatMed, 2011. 17(10): p. 1225-7.). Under a Cultrex® Organoid Qualified BME,Type 2 designation, several formulations of Cultrex® BME are describedfor organoid culture including Cultrex® Basement Membrane Extract, Type2, PathClear® (provided as part of a protocol for subculturing normalhuman gastric organoids which was derived from the submerged method asdescribed in Barker, et al., Lgr5+ve Stem Cells Drive Self-Renewal inthe Stomach and Build Long-Lived Gastric Units In Vitro. Cell Stem Cell,2010. 6(1): p. 25-36)) and Cultrex® Reduced Growth Factor BasementMembrane Extract, Type 2, PathClear® (Human Colorectal Cancer (CRC)organoids grown from single cells on Cultrex® BME Type 2 Reduced GrowthFactor). Additional products that might find use include but are notlimited to Cultrex® 3-D Culture Matrix® Reduced Growth Factor BasementMembrane Extract, PathClear® (allowing for the formation of acinar andother hollow unnamed structures in vitro); Cultrex® Basement MembraneExtract, PathClear®; Cultrex® Stem Cell Qualified Reduced Growth FactorBasement Membrane Extract, PathClear®; Cultrex® Basement MembraneExtract, Type 3, PathClear®. The PathClear® designation means that inaddition to standard sterility, endotoxin and MAP testing, the basementmembrane extract is tested by PCR and is clear of 31 pathogens andviruses, including lactate dehydrogenase elevating virus (LDEV).Cultrex® BME Type 2 provides a formulation with a higher in tensilestrength when compared to the original BME, while Cultrex® BME Type 3 isphysiologically aligned with the in vivo solid tumors environment and isrecommended for xenografts and other in vivo applications.

EXPERIMENTAL

The following is a summary of contemplated experimental strategy. Withthe experimental strategy as delineated herein, we aim to develop in astepwise approach a number of distinct although interlocking platformsto study human cancer as delineated below. Proof of concept in all ofthe following steps will be provided by identical experiments in chipspopulated with cells from transgenic/humanized mouse models amenable topurification and real-time tracing of all the relevant cells to be used,together with well-characterized tumor cells.

Example 1 Tumor Development and Expansion within the Normal Tissue

Seeding Cancer-On-Chips with cells derived from fresh human tumorspecimens demonstrating grow in the Chip and providing a populationcomposition representative of the original tumor, i.e. confirm we do notselect from the more aggressive or the more differentiated cell type.Endothelial cells will be incorporated, the interaction between tumorcells and endothelial cells will be characterized and angiogenesis willbe monitored. In this experiment, compounds targeting the cell cycle orspecific cell functions such as autophagy, as well as tumor vasculatureand angiogenesis could be tested for determining whether knownanticancer agents have similar effects in Cancer-On-Chips, in additionto testing compounds as potential therapeutics.

Example 2 Tumor Invading in the Surrounding Tissues, i.e. the Stroma andthe Supporting Endothelial and Lymphatic System

In this example, the response of stroma to tumor cells and the effectsof activated (tumor-derived) fibroblasts on tumor cells biology areshown. Activation of stroma, interaction with tumor cells, and changesin the phenotype of tumor cells following interaction with stroma,effects on the vascular systems such as changes in permeability,neovascularization, metabolic function will be evaluated.

Example 3 Incorporation of the Immune System in the Tumor andStroma-On-Chip

In this example, antigen priming and/or immune cell migration will bestudied in relation to cancer cell/tumor growth. In one embodiment,dendritic cells (DC) are fluidically circulated through the tumorcompartment in order to prime them to tumor antigens. In one embodiment,resident immune cells are included to study their role in cancerdevelopment. In one embodiment, a separately maintained culture ofcytotoxic cells dyed with a cell dye to be distinct and traceable bymicroscopy, will be exposed to the primed DC to assess thephysiologically relevant interaction and then will be flown (fluidicallycirculated) in the system through the vascular system to assess theexistence of an in vivo relevant “immune privileged” environment. In oneembodiment, a model for recruitment of immune cells by the tumor will beevaluated where either immune cells have migrated within the tumor tobecome a critical component of the immunosuppressive properties of thetumor, such as myeloid suppressor cells (MSC), or surround the tumor, orare excluded from entry (such as CD8+ T cytotoxic cells). In oneembodiment, immune cells that move away will be collected with theeffluent from the Cancer-In-Chip.

Example 4 Tumor and Stroma and Immune System-On-Chip

In this example, Mesenchymal stem cells (MSCs) will be studied inrelation to cancer cell/tumor growth. In one embodiment, recruited MSCs,originating from Bone marrow-Chip, i.e. a microengineered model thatreplicates native niche and key immunological function of mammalian,e.g. human bone marrow in vivo, will be studied in relation to cancercell/tumor growth.

This Bone marrow-Chip in communication with a Cancer-On-Chip will beconstructed by incorporating human bone marrow obtained surgically fromthoracectomy into a perfusable microfluidic device that contains avascularized three-dimensional tissue culture scaffold. The design ofthis model will enable spontaneous anastomosis of the microvasculaturein the marrow with a network of microengineered blood vessels, making itpossible to generate and precisely control vascular perfusion of thebone marrow. Once this culture is established, human hematopoietic stemcells will be introduced into the engineered tissue and induced todifferentiate into the myeloid lineage. Functional validation of thismodel will be achieved by measuring mobilization of neutrophils inresponse to colony-stimulating factors such as G-CSF or CXC chemokinessuch as IL-8.

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The invention claimed is:
 1. A method comprising: 1) providing a) a microfluidic device comprising: a body having a first channel and a first chamber, an at least partially porous membrane positioned at an interface region between the first channel and the first chamber, the membrane comprising a top surface and a bottom surface, said top surface facing the first chamber; living epithelial cells disposed within the first chamber as a layer of cells; and living tumor cells in contact with said living epithelial cells, said tumor cells originating from a biopsy, and disposed within at least one of the first chamber or the first channel; and 2) testing said epithelial cell layer for invasion by said tumor cells.
 2. The method of claim 1, wherein the first chamber comprises a second channel.
 3. The method of claim 1, wherein the first chamber comprises an open region.
 4. The method of claim 1, wherein said tumor cells comprise melanoma cells.
 5. The method of claim 1, wherein the method further comprises endothelial cells disposed within the first channel.
 6. A method comprising: 1) providing a) a microfluidic device comprising a body having a first chamber, an at least partially porous membrane comprising a top surface and a bottom surface, said top surface facing the first chamber; b) living epithelial cells disposed within the first chamber as a layer of cells; and c) living tumor cells in contact with said living epithelial cells, and disposed within said first chamber; and 2) testing said epithelial cell layer for invasion by said tumor cells.
 7. The method of claim 6, wherein said tumor cells comprise melanoma cells. 